Documentation of Indigenous Antiparasitic Practices and Scientific Evaluation of Some Ethnobotanicals for Their Anthelmintic Activity

DOCUMENTATION OF INDIGENOUS ANTIPARASITIC PRACTICES AND SCIENTIFIC EVALUATION OF SOME ETHNOBOTANICALS FOR THEIR ANTHELMINTIC ACTIVITY

Nadeem Badar Reg. # 91-ag-750

A Thesis Submitted in Partial Fulfillment of the Requirement for the Degree of

DOCTOR OF PHILOSOPHY

PARASITOLOGY

FACULTY OF VETERINARY SCIENCE UNIVERSITY OF AGRICULTURE FAISALABAD, PAKISTAN 2011

DECLARATION

I hereby declare that contents of the thesis, “Documentation of indigenous antiparasitic practices and scientific evaluation of some ethnobotanicals for their anthelmintic activity” are product of my own research and no part has been copied from any published source (except the references, some standard mathematical or genetic models/ equations/protocols etc.). I further declare that this work has not been submitted for award of any other diploma/degree. The university may take action if the information provided is found inaccurate at any stage. (In case of any default the scholar will be proceeded against as per HEC plagiarism policy).

______Signature of the Student Name: Nadeem Badar Reg. No. 91-ag-750

ACKNOWLEDGEMENT

First of all I am thankful to my Allah Who enabled me to collect and to

contribute my findings in the existing scientific knowledge. Then I record my

deep sense of gratitude and appreciation to Prof. Dr. Zafar Iqbal,

Department of Parasitology, University of Agriculture, Faisalabad for his

constant encouragement and inspiring guidance throughout my postgraduate

period. I have no appropriate words that fully convey the sense of immense

thanks and deep gratitude that I owe to my respectable committee members

Prof. Dr. Muhammad Nisar Khan, Chairman, Department of Parasitology,

University of Agriculture, Faisalabad and Prof. Dr. Muhammad Shoaib Akhtar

for their kind supervision and affections. I am grateful to the Lab. Attendants

and those who co-operated with me during data collection.

I can not forget the prayers and encouragement of my affectionate

parents who always supported me throughout my life and I am also thankful to

the higher education commission (HEC) of Pakistan for partial funding of this

project.

Nadeem Badar

CONTENTS

Chapter No. Contents Page No.

LIST OF TABLES i

FIGURE iii

APPENDIX iii

1 INTRODUCTION 1

2 REVIEW OF LITERATURE 3

3 MATERIALS AND METHODS 18

4 RESULTS 27

5 DISCUSSION 54

6 SUMMARY 73

7 REFERENCES 75

LIST OF TABLES

Table No. Content Page No. 1 Plants used as anthelmintics 4 2 Plants used as antiprotozoals 9 3 Plants used as insecticides 12 4 Plants used as acaricides 14 5 Tests/assays for scientific activity evaluation of different 16 ethnobotanicals 6 Plants selected for anthelmintic activity 22 7 Layout plan for crude powder and crude aqueous methanolic extract of 25 Acacia nilotica (Bark & Leaves), Vitex negundo (seeds) and Arundo donax (leaves) given to different groups of sheep naturally infected with mixed species of gastrointestinal nematodes 8 Layout plan for crude powder and crude aqueous methanolic extract of 25 Amomum subulatum (Fruit) given to different groups of sheep naturally infected with mixed species of gastrointestinal nematodes 9 Layout plan for crude powder and crude aqueous methanolic extract of 26 Areca catechu (seeds) and Ferula assa-foetida (latex) given to different groups of sheep naturally infected with mixed species of gastrointestinal nematodes 10 Botanical, local and English names of the plants documented from 27 district Jhang (Punjab, Pakistan) for their use in ethnoveterinary medicine 11 Name of plants, representing families and frequency of their usage in 28 ethnoveterinary medicine in district Jhang (Punjab, Pakistan) 12 Top ten most frequently reported plants for their usage in 29 ethnoveterinary medicine in Jhang (Punjab, Pakistan) 13 Inventory of plants used for the treatment of different diseases/ 30 conditions of livestock reported by the local respondents in Jhang (Punjab, Pakistan) 14 Number and nature of EVM practices for different diseases/conditions 35 documented from Jhang (Punjab, Pakistan) 15 Plants, diversity of their usage and contribution in total number of 36 EVM practices for different diseases/conditions of livestock in Jhang (Punjab, Pakistan) 16 In vitro effect of crude aqueous methanol extracts of different plants on 39 survival of H. contortus of sheep in comparison with Levamisole 17 In vitro effect of different fractions of crude aqueous methanol extracts 40 of Ferula assa-foetida on survival of H. contortus of sheep in comparison with Levamisole

i LIST OF TABLES…continued

Table No. Content Page No. 18 In vitro effect of different fractions of crude aqueous methanol extracts 41 of Acacia nilotica bark on survival of H. contortus of sheep in comparison with Levamisole 19 In vitro effect of different fractions of crude aqueous methanol extracts 42 of Areca catechu on survival of H. contortus of sheep in comparison with Levamisole 20 In vitro effect of different fractions of crude aqueous methanol extracts 43 of Amomum subulatum on survival of H. contortus of sheep in comparison with Levamisole 21 In vitro effect of different fractions of crude aqueous methanol extracts 44 of Vitex negundo on survival of H. contortus of sheep in comparison with Levamisole 22 In vitro effect of different fractions of crude aqueous methanol extracts 45 of Acacia nilotica leaves on survival of H. contortus of sheep in comparison with Levamisole 23 In vitro effect of different fractions of crude aqueous methanol extracts 46 of Arundo donax on survival of H. contortus of sheep in comparison with Levamisole 24 Ranking of efficacy of different fractions of plants against H. contortus 46

25 Percent egg hatch and LC50 of crude aqueous methanol extracts and 47 fractions of different plants 26 Top 10 highly ovicidal plant crude aqueous methanol extracts and/or 48 fractions in comparison with control 27 Regression values and correlation of regression of the effect of 49 different plants on egg hatching 28 Top 10 plant crude aqueous methanol extracts and/or fractions having 50 best dose dependant ovicidal effects 29 Effect of crude powder and crude methanol extracts of different plants 51 on eggs per gram of feces (Mean±SEM) in sheep naturally infected with mixed species of gastrointestinal nematodes in comparison with untreated and levamisole treated animals 30 Efficacy ranking based on reduction in faucal egg counts as on day 12 52 post-treatment (PT) of crude powder and crude methanol extract of different plants 31 Ranking of efficacy of different CAME/fractions of plants based on 53 three tests employed in the study

FIGURE

Fig. No. Content Page No.

1 Map of Pakistan showing the study area 19

APPENDIX

Appendix Content Page No. No.

1 List of private Livestock farms included in the study 25

iii Chapter # 1 Introduction Livestock production depends on the feed supplies, good health coverage and appropriate animal husbandry practices. Many climatic and casual factors are experiencing high degree of parasitic attack and plague on them resulting in their decreased longevity, survivorship, infertility and productivity. The productive and reproductive potential of domestic livestock is adversely affected because of clinical and sub-clinical infections. All the ailments afflicting animals are counterproductive. Gastrointestinal helminthiasis (especially nematodosis) is, however, of high economic significance in view of its insidious nature and easy transmissibility due to under feeding, availability of a wide variety of hosts, vectors, inadequate/low level of awareness and animal health cover. For centuries, most of the human and animal population in Indo-Pakistan subcontinent has relied on a system of traditional medicine. The traditional medicine system prevailing in this area is also termed as Eastern Medicine, Unani Medicine, Islamic Medicine or Ethno-medicine. Ethno-veterinary medicine (EVM) is the traditional system of maintaining animal health and curing diseases of animals that is based on folk beliefs and traditional knowledge, skills, methods and practices (Mathias-Mundy and McCorkle, 1989). EVM is a traditional system that local people, through trial-and-error and also deliberate experimentation, developed to keep their animals healthy and productive. Though, this old method of treatment has been replaced by allopathy in some parts of globe (Tabuti et al., 2003), yet it is contributing significantly in improving the animal health in developing countries. It can play a very important role in sustainable veterinary medicine in modern world (Lin et al., 2003), especially in Indian subcontinent due to various benefits associated with this method of treatment, e.g. lower cost, greater accessibility and apparent efficacy (Mwale et al., 2005). Tradition of EVM is very old in Indo-Pakistan region and it is evident from its documentation in the form of Rig-veda (from 4500 to 1600 BC) and Ayurveda from 2500 to 600 BC (from Zaman and Khan, 1972; Somvanshi, 2006). Old books and scriptures of 2350 BC like; Agni Purana, Skanda Purana, Matsya Purana, Devi Purana, Garuda Purana and Linga Purana describes the documentation of phytotherapy for treatment of different diseases of animals (Somvanshi, 2006). Majority of the EVM surveys and validation studies indicate much wider and effective use of plants as anthelmintics compared with other diseases/conditions (Iqbal et al., 2004, 2005, 2006, 2006a,b,c; Jabbar et al., 2007; Hussain et al., 2008). The tremendous use of plants as anthelmintics for the treatment of helminthiasis is attributed to its high prevalence and heavy

1 production losses in third world countries (FAO, 1974; Dhar et al., 1982; Ibrahim et al., 1984; Ashraf, 1985; Waller, 1987; Sykes, 1994; Kochapakdee et al., 1995; Perry and Randolph, 1999), for example, Pakistan (Iqbal et al., 1993; Qayyum, 1996). Furthermore, development of anthelmintic resistance (Waller and Prichard, 1985) in nematodes against almost all anthelmintic groups, commonly being used in these countries and problem of chemical residues in animal products (Kaemmerer and Butenkotter, 1973) have also motivated scientists from developed countries to screen the medicinal plants for their anthelmintic activity. Indigenous knowledge of cure and treatment of animals using medicinal plants and other products has passed on from one generation to next and it greatly varies from one region to the other, depending upon availability of local plants and ethnic groups living in a particular area. In Pakistan, a large variety of medicinal plants is abundantly available, and most of them are being used by local healers for therapeutic purposes without specific knowledge of their active ingredients. There is an urgent need to document this important knowledge in different areas of Pakistan, before its disappearance due to rapid socio-economic, technological and environmental changes. To date, very little work has been done on documentation of EVM in Pakistan (Jabbar et al., 2007; Hussain et al., 2008; Dilshad et al., 2008) in contrast to other countries, like; India (Savithramma et al., 2007), Tanzania (Sheila et al., 2007), Brazil (Albuquerque et al., 2007), Nigeria (Kola et al., 2008), Ethiopia (Wondimu et al., 2007) and Indonesia (Mahyar et al., 1991; Katrin et al., 2008). Above considerations led to the development of proposal for this study. The objectives of the present study were to (i) document the ethno-veterinary practices with particular reference to the antiparasitic practices in some selected parts of District Jhang (Pakistan), and (ii) validate the claims of local healers regarding anthelmintic activity of some plants in animals.

2 Chapter # 2 Review of Literature As stated by Schillhorn van Veen (1997), most of the history of animal husbandry, farmers and herders has relied on traditionally derived practices in management and health. Origin of the modern veterinary medicine can be traced in traditional veterinary medicine of India, Middle East and China. For example in 1800 BC King Hammurabi of Babylon laid out laws concerning the veterinarian’s fee for treating cattle and donkeys. Records about animal health can be found in the Rock Edict II of King Ashoka (269-232 BC) because cattle and other animals were worshiped by early Buddhist in Indian region. It has been reported that hospitals for animals were established in this era, and later Egypt, Arabia and China developed into centers of veterinary knowledge (quoted by Lodrick, 1981). Globally, old age men are the custodians of EVM knowledge. Unfortunately, however, traditional knowledge of medicinal plants is vanishing due to loss of these octogenarians, dying with their knowledge unrecorded (Cox, 2000). These octogenarians were self made botanist due to deep knowledge of their ecosystems, who could name virtually every plant found on their land. In this backdrop, documentation of EVM is helpful in understanding its scientific rationale, growth of new concepts and adaptation percent of modern technologies. It is also of great importance in increasing awareness among the youngsters, developing appreciation for the traditional knowledge and renewing pride among the farming community and identifying the medicinal plants (Makhubu, 1998; Cox, 2000). EVM is helpful in animal health care and maintenance of the productivity of animals, which has evolved through observation, trial and error, perfecting the techniques based on the experiences gathered through experimentation, and handling the resulting information down from one generation to the next (McCorkle, 1995). Over centuries, people have developed alternative ways to treat different ailments of animals due to high cost of synthetic drugs, inaccessibility, and side effects associated with modern drugs (Kumar, 2002). Unfortunately, however, EVM practices are scarcely documented and much of this valuable knowledge has lost, as it passes from one generation to the other by verbal communication (Mathias, 2001). That is why there is an inherent danger of losing this valuable veterinary knowledge. Documentation also provides country's geographical indications and rights thereupon for avoiding patent debate. Traditional bioprospecting provide the basis for ethno medicine and ethno veterinary practices (Ole-Miaron, 1997), which are an integral part of people’s culture (Brandt et al., 1995). For a very long time, modern bioprospecting, which depends on scientific

3 analysis, has preyed upon traditional bioprospecting to benefit the pharmaceutical industry (Ole- Miaron, 2003). Farming community is using both (effective products and mythological religious faiths) for curing various diseases. It has been observed that veterinarians are aware of EVM, but they do not recommend/use this knowledge because of the lack of scientific validation. In the light of objectives given in the introduction, literature on the documentation and/or validation of traditional anti-parasitic practices in different parts of the world has been selectively reviewed in this chapter. EVM is mainly constituted by plants; therefore, main focus of this review is on the use of plants as anthelmintics, antiprotozoals and acaricides. 2.1. Plants used as anthelmintics Numerous plants have been reported in the literature for treatment of parasitic diseases of animals and humans (Nadkarni, 1954; Chopra et al., 1956; Said, 1969; Akhtar et al., 2000). Earlier, in vitro anthelmintic activity of plants was reported on the basis of their toxic effects on earthworm, Pheretima posthuma (Siddiqui and Garg, 1990; Garg and Siddiqui, 1992). Later, researchers started evaluating anthelmintic activity of plants against parasitic worms like; hookworms, Haemonchus (H.) contortus, tapeworms and/or A. lumbricoides, in vitro (Kalyani et al., 1989; Nakhare and Garg, 1991; Garg and Siddiqui, 1992; Garg and Jain, 1992). In vivo trials are also conducted for the evaluation of anthelmintic activity of plant extracts/parts. The parameters for such an activity included expulsion of worms from their hosts (Asuzu and Onu, 1994; Desta, 1995) or reduction in the number of eggs per gram of feces (EPG) passed by the infected hosts compared with animals treated with commercial anthelmintics (Akhtar, 1988). Anthelmintic activity of some plants has been reviewed by Akhtar et al. (2000). Such an activity has also been reported for some plants like that of sorghum extract (Iqbal et al., 2001), Zingiber officinale, Allium sativum, Ficus religiosa, and Cucurbita mexicana (Iqbal et al., 2001a), Artemisia brevifolia (Iqbal et al., 2004), Calotropis procera (Iqbal et al., 2005), Nicotiana tabacum (Iqbal et al., 2006a) and Butea monosperma (Iqbal et al., 2006b). The anthelmintic activity of different plants reported in literature has been tabulated in Table 1. Table 1: Plants used as anthelmintics

Plant Part used Helminth (s)/Remarks Animal species Reference (s) Annona senegalensis Leaf, bark, root Nippostrongylus (N.) Rat Ibrahim et al., 1984 brasiliensis Acacia albida Seeds Worm infestation Sheep, goat Nwude and Ibrahim, 1980 Adhatoda vesica Roots Mixed GI nematodes Sheep Lateef et al., 2003 Agati gratifola Not reported Ascaris (A.) lumbricoides Human Kalesaraj, 1974 Ageratum conyzoides Leaves and Tapeworms Not reported Sharma et al., 1979 flowers Aglaia odorattissima Root bark Earthworms Not reported Nanda et al., 1987 Agrimonia eupatori Not reported Anthelmintic Humans Farnsworth et al., 1985 Agrimonia pilosa Agrimophol Tape worms Not reported Xiao and Lin, 1986 Alangium lamarckii Root-bark Ascarids Poultry Dubey and Gupta, 1969 Alangium larmarckii Root bark Hookworms, ascarids Dogs, poultry Dubey and Gupta, 1968

4 Table 1 Contd. Plant Part used Helminth (s)/Remarks Animal species Reference (s) Albizia anthelmintica Bark Anthelmintic Cattle, goat, sheep Minja, 1989; ITDG and IIRR, 1996 Root Fasciolosis Cattle, goat, sheep Bark Lungworms Camel Albizia coriavera Bark Fasciolosis, lungworms Cattle, goat, sheep ITDG and IIRR, 1996 Albizia lebbek Bark A. lumbricoides In vitro Kalesaraj, 1975 Allium sativum Bulb Roundworms Cattle, goat, sheep ITDG and IIRR, 1996; Iqbal et al., 2001a Bulb Ascaridia (As.) galli Chicken Das and Thakuria, 1974 Aloe barteri Bulb A. lumbricoides In vitro, Kalesaraj, 1975 Leaves Nippostrongylus spp. Rat Ibrahim et al., 1984 Alpinia calcarata Rhizomes A. lumbricoides In vitro Kalesaraj, 1975 Cucuruma aromatica Ammora wallichii Stem As. galli In vitro Kaushik et al., 1981 Calamintha umbrosa Whole plant Picus religiosa Stem and bark Sentia myrtina Whole plant Sumplocos Leaves crataegoides Amomum Root and As. galli In vitro Kaushik et al., 1981 aromaticum Rhizome Anacardium Not reported Earthworms, tapeworms Not reported Garg and Kasera, 1982, 1982a occidentale Ananas comosus Fruit As. galli Chicken Fernandez, 1991 Ananas sativus Not reported Taenia species and In vitro Neogi et al., 1964 Paramphistomum (P.) cervi Ananas sativus Not reported Earthworms In vitro Chakraborty et al., 1976 Annona cherimolia Nippostrongylus spp. Rat Bories et al., 1991 Annona muricata Annona braziliensis Molinema dessetae Anogeissus Bark, seed N. brasiliensis Rat Ibrahim et al., 1984 leiocarpus Anogeissus Bark Anthelmintic In vivo Bizimana, 1994 leiocarpus Securinega virosa Leaves and stem Khaya senegalansis Bark Nauclea latifolia Roots Anthocephalus Stem and Bark As. galli In vitro Kaushik et al., 1981 indicus Areca catechu Nut Taenicidal Cattle, goat, dog Roepke, 1996 Areca catechu Dried ripe seeds Tape worms Dogs, poultry British Veterinary Codex, 1953 Artabotrys Leaves Pheretima posthuma, Taenia In vitro Siddiqui and Garg, 1990 odoratissimus (T.) solium and A. lumbricoides Artemisia abrotanum Not reported Anthelmintic Not reported Krause, 1993 Artemisia absinthium Not reported Anthelmintic Not reported Francois, 1974; Bara et al., 1999; Guarrera, 1999 Artemisia annua Not reported Schistosoma (S.) mansoni Hamster, mice Shuhua et al., 2000 Artemisia brevifolia Not reported H. contortus Sheep Iqbal et al., 2004 Artemisia herba-alba Shoots H. contortus Goat Idris et al., 1982 Artemisia Leaves A. suum Pig (in vitro) Slepnev, 1970 inflorescence Artemisia maritima Whole plant Anthelmintic Not reported Krantz and Carr, 1967; Narayana et al., 1976; Akhtar, 1984; Sharma, 1993; Hammond et al., 1997 Whole plant Neoascaris vitulorum Buffalo calves Akhtar et al., 1982; Farnsworth et al., 1985; Sherif et al., 1987; Fernandez, 1991 Artemisia Flavonoids and Anthelmintic Not reported Holeman et al., 1991 mesatlantica sesquiterpene lactones Artemisia Not reported Anthelmintic Not reported Abu-Niaaj et al., 1996 monosperma Artemisia pallens Not reported Anthelmintic Not reported Anonymous, 1956; Nakhare and Garg, 1991 Artemisia scoparia Not reported Anthelmintic Not reported Naqvi et al., 1991 Artemisia senna Not reported Anthelmintic, Cestodes Canine Francois, 1974; Narayana et al., 1976 Azadirachta indica Cake and Anthelmintic Small ruminants Mostofa et al., 1996; Gowda, 1997 Leaves

5 Table 1 Contd. Plant Part used Helminth (s)/Remarks Animal species Reference (s) Azadirachta indica Seeds H. contortus, Trichostrongylus Lambs Hördegen et al., 2003 (T.) colubriformis Melia azedarach Seeds Ananas comosus Leaves Vernonia Seeds anthelmintica Embelia ribes Fruits Fumaria parviflora Whole plants Caesalpinia crista Seeds Bixa orellana Seeds As. galli, A. suum Chicken, Pig Fernandez, 1991 Boswellia dalzelii Bark Anthelmintic Sheep, goat Nwude and Ibrahim, 1980 Boswellia serrata Not reported Earthworms, tapeworms In vitro Girgune et al., 1978 Buddlea asiatica Not reported Earthworms, tapeworms, Not reported Dengre, 1982 hookworms Butea frondosa Seeds Anthelmintic, As. galli, A. Chicken (In vitro), Kalesaraj and Kurup, 1962, 1968; Joshi, lumbricoides canine, human 1970; Narayana et al., 1976; Lal et al., 1976, 1978; Shilaskar and Parashar, 1989 Butea frondosa Not reported Oxyurids Mice Mehta and Parashar, 1966 Butea frondosa Seeds As. galli In vitro Lal et al., 1976 Butea monosperma Seeds Anthelmintic, GI nematodes Sheep and others Kalesaraj and Kurup, 1968; Chandra and Sabir, 1978; Lal et al., 1978; Prashanth et al., 2001; Iqbal et al., 2006b Butea superba Not reported Anthelmintic Not reported Charka, 1948; Chopra et al., 1958 Caesalpina crista Seeds Toxocara (T.) vitulorum, Buffalo calves, Akhtar et al., 1985 As. galli chicken Javed et al., 1994 Seeds H. contortus Sheep, goat (In vitro) Sharma et al., 1971 Calliandra Legume H. contortus, Sheep Parker and Palmer, 1991 calothyrsus Trichostrongylus, Strongyloides papillosus Calliandra Root T. canis, GI nematodes, Dog Adewunmi and Akubue, 1981 portoricensis Leaves, H. contortus Sheep Garg and Atal, 1963; Jain et al., 1996; Al- Flowers Qarawi et al., 2001; Iqbal et al., 2005 Calotropis procera Capillipedium Oil Pheretima posthuma, T. In vitro Siddiqui and Garg, 1990 foetidum Grass solium and A. lumbricoides Cymbopogon martini Carica papaya Seeds A. lumbricoides, As. galli Human, Chicken Dhar et al., 1965; Lal et al., 1976 Latex from As. galli, A. suum, Chicken, Pig, Mice Mursof and He, 1991; Satrija et al., 1994; Fruit Heligmosomoides polygyrus Satrija et al., 1995 Carissa edulis Roots Roundworms Cattle, goat, sheep ITDG and IIRR, 1996 Carum copticum Seeds A. lumbricoides Human Krantz and Carr, 1967; Kalesaraj, 1974 Cassia alata Seeds As. galli Chicken Fernandez, 1991 Cassia occidentalis Leaf N. brasiliensis Rat Ibrahim et al., 1984 Cassia spectalis Root Roundworms Cattle, goat, sheep ITDG and IIRR, 1996 Chebulic Not reported Anthelmintic activity Not reported Gaind et al., 1964 myrobalans Belleric myrobalans Emblic myrobalans Chenopodium album Leaf Nematode Sheep Akhtar et al., 1999 Chenopodium spp. Oil Ascaris spp., Toxascaris, Horses, pigs, dogs British Veterinary Codex, 1953, 1965 Toxocara, Strongylus spp. Chloroxylon Oil Earthworms, tapeworms, Not reported Dengre, 1982 swientenia hookworms Chrysanthemum spp. Not reported H. contortus Chicken Rebrassier, 1934 Chrysophyllum Stem H. contortus Cattle Fernandez, 1991 cainito Cinnamomum Oil Earthworms, tapeworms In vitro Girgune et al., 1978 tamala Cissampelos Root Anthelmintic Not reported Minja, 1989 mucromata Citrus acida Rind A. lumbricoides In vitro Kalesaraj, 1975 Citrus aromatica Citrus medica Combretum Root Guineaworm Humans Sofowora, 1982 mucronatum Commiphora mukul Oleo-gum resin Tapeworms, hookworms Not reported Kakrani and Kalyani, 1984 Croton macrostachys Leaves Anthelmintic Not reported Minja, 1989

6 Table 1 Contd. Plant Part used Helminth (s)/Remarks Animal species Reference (s) Cucurbita mexicana Seeds Moniezia expansa, Not reported Shrivastava and Singh, 1967 Fasciolopsis (F.) buski, A. lumbricoides, Hymenolepis (H.) diminuta Cucurbita moschata Seeds Cestode Human Xiao and Lin, 1986 Cucurbita pepo Not reported H. contortus (mature) Goat (in vitro) Sharma et al., 1971 Momordica charantia Cyathocline lyrata Essential oil Tapeworms, hookworms In vitro Shrivastava, 1979 Cymbopogon nardus Essential oil Earthworms In vitro Kokate and Varma, 1971 Cymbopogon citrates Cyperus rotendus Not reported Tapeworms, earthworms Not reported Girgune et al., 1979 Datura quercifolia Fruit As. galli In vitro Kaushik et al., 1981 Datura metal Diospyros mollis Diospyrol Necator (N.) americanus Golden hamster, Sen et al., 1974 Nematodirus (N.) dubius, H. Mice nana Diospyrol N. americanus Golen hamster Sen et al., 1974 Diospyrol N. dubtus, H. nana Mice Sen et al., 1974 Diospyrus scabra Seeds Fasciolosis, lungworms Cattle, goat, sheep, ITDG and IIRR, 1996 camel Dodonea viscosa Leaves Intestinal worms Not reported Sharma and Singh, 1989 Dryopteris filixmas Male fern Moniezia, tape womrs, Not reported British Veterinary Codex, 1953 Dirocoelium, Fasciola Embelia Root Anthelmintic Not reported Minja, 1989 kilimandschiraca Embelia schimperi Seed, root, Fruit Anthelmintic, H. diminuta Rat Bøgh et al., 1996 Embellia ribes Not reported Mixed GI nematodes Ruminants Chopra et al., 1956; Ikram and Hussain, 1978 Fruit Taenia species, P. cervi, GI Goats Neogi et al., 1964; Javed and Akhtar, nematodes 1990 Embellia ribes Seeds Tape worms Poultry Qureshi and Sabir, 1979 Erythrina Bark Fasciolosis Ruminants Nwude and Ibrahim, 1980 senegalensis Eupatorium Flowers A. lumbricoides and T. solium Not reported Garg and Nakhare, 1993 triplinerve Evodia rutaecarpa Not reported Ascarids, Ostertagia Pig (in vitro) Perrett and Whitfield, 1995 circumcincta Sheep (in vitro) Ferula foetidissima Not reported Haemonchus, Bunostomum, Sheep Pustovoi, 1968 Chabertia,Nematodirus Ficus religiosa Not reported Anthelmintic In vitro Iqbal et al., 2001a Flemingia vestita Root-tuber peel Raillietina echinobothrida Domestic fowl (in Pal and Tandon, 1998 vitro) Flemingia vestita Root-tuber peel F. buski Pig (in vitro) Kar et al., 2002 Fumaria parviflora Plant powder Trichostrongylus, Sheep, buffalo Akhtar and Javed, 1985; Kailani et al., Haemonchus, Trichuris spp., 1995 Fasciola spp. Gardenia lucida Essential oil Tapeworms, earthworms Not reported Girgune et al., 1979 Hagenia abyssainicia Fruit Roundworms Cattle, goat, sheep ITDG and IIRR, 1996 Hedychium Rhizomes Earthworms, tapeworms Not reported Dixit and Varma, 1975 coronarium Hedychium spicatum Helleborus niger Stem A. lumbricoides Human Kalesaraj, 1974 Heracleum sosnoskyi Not reported Strongylosis, GI nematodes Sheep Gadzhiev and Eminove, 1986, 1986a Hyoscyamus niger Seeds Mixed GI nematodes In vivo Akhtar and Ahmad, 1990 Inula racemosa Essential oils Earthworms, tapeworms Not reported Mishra et al., 1979 Juglans regia Not reported H. contortus Goat, in vitro Sharma et al., 1971 Musa paradisaca Scindapsus officinalis Khaya senegalansis Bark Fasciola spp. Not reported Bizimana, 1994 Lagenaria siceraria Seeds Cestodes, Moniezia, Avitellina Sheep Akhtar and Riffat, 1987 spp. Lansium domesticum Seeds As. galli Chicken Fernandez, 1991 A. suum Pig H. contortus Goat Lantana trifolia Fruit Fasciolosis, lungworms Cattle, goat, sheep ITDG and IIRR, 1996 Lantana camara var. Seeds Anthelmintic activity Not reported Avadhoot et al., 1980 aculeate

7 Table 1 Contd.. Plant Part used Helminth (s)/Remarks Animal species Reference (s) Lawsonia inermis Leaf Fasciolosis Sheep, goat Nwude and Ibrahim, 1980 Leucaena Seeds As. galli Chicken Fernandez, 1991 leucocephala A. suum Pig H. contortus Goat Limnophila conferta Not reported Anthelmintic activity Not reported Reddy et al., 1991 Litsea chinensis Not reported Earthworms, Tapeworms Not reported Mishra et al., 1979 Macuna prurita Not reported Taenia species, P. cervi Not reported Neogi et al., 1964 Mallotus Fruit powder GI cestodes Beetal goats Akhtar and Ahmad, 1992 Fruit Tape worms Not reported British Veterinary Codex, 1953 Mangifera indica Seed A. lumbricoides Human Kalesaraj, 1974 Matteuccia orientalis Root Fasciola spp. Cattle Shiramizu et al., 1993 Melia azedarach Fruit, leaf Taenia species, P. cervi, H. In vitro Neogi et al., 1964; Sangwan and contortus Sangwan, 1998 Fruit As. galli Chicken Akhtar and Riffat, 1985a Fruit Haemonchus, Goat Akhtar and Riffat, 1984 Trichostrongylus, Trichuris, Chabertia spp. Melia toosendan Not reported Ascarids Not reported Xiao and Lin, 1986 Mimosa pudica Stem H. contortus Not reported Fernandez, 1991 Mitragyna stipulosa Root Guineaworm Humans Sofowora, 1982 Momordica Not reported As. galli In vitro Lal et al., 1976 charantia Stem A. suum Pig Fernandez ,1991; Farnsworth et al., 1985 As. galli Chicken H. contortus Goat As. galli Chicken Moringa oleifera Seeds A. suum Pig Fernandez, 1991 H. contortus Goat H. contortus Goat Roots Mixed GI nematodes Sheep Akhtar and Ahmad, 1990 Myrsine africana Leaf Roundworms Cattle, goat, sheep ITDG and IIRR, 1996 Nicotiana tabacm Nicotine Moneizia, Ascaridia, Not reported British Veterinary Codex, 1953, 1965 sulphate Cooperia, Haemonchus, Nematodirus, Ostertagia, Trichostrogylus spp. Nigella sativa Seeds Antifasciolic Bufffalo Kailani et al., 1995 Peganum harmala Seeds Mixed GI nematodes, cestode Goat Akhtar and Ahmad, 1991 infection Peganum harmala Seed GI cestodes Goat Akhtar and Riffat, 1986 Piper betle Not reported Earthworms In vitro Ali and Mehta, 1970 Psitacia integrrima Seeds Earthworms, tapeworms Not reported Mishra et al., 1979 Psoralea coylifolia Seed powder GI nematodes Sheep Javed and Akhtar, 1986 Punica granatum Fruit-rinds GI nematodes, cestodes Sheep Akhtar and Riffat, 1985 Not reported A. lumbricoides In vitro Kalesaraj, 1975 Not reported H. contortus In vitro Prakash et al., 1980 Quisqualis indica Stem A. suum, H. contortus Goat Farnsworth et al., 1985 A. suum Pig Fernandez, 1991; Farnsworth et al., 1985 As. galli Chicken H. contortus Goat Quisqualis indica Seeds Ascaris spp. Not reported Xiao and Lin, 1986 Randia dumetorum Seeds Earthworms, tapeworms Not reported Mishra et al., 1979 Rapanea Seeds Roundworms Cattle, Goat, Sheep ITDG and IIRR, 1996 melanoploeos Rhamnus principides Leaves Anthelmintic Not reported Minja, 1989 Rhus vulgaris Root Roundworms Cattle, goat, sheep ITDG and IIRR, 1996 Sapindus trifoliatum Not reported As. galli In vitro Lal et al., 1976 Saussurea lappa Root Mixed species of nematodes Sheep Akhtar and Hassan, 1985 Buffalo-calves Akhtar and Makhdoom, 1988 Semecarpus Nuts Anthelmintic Not reported Chattopadhyaya and Khare, 1969 anacardium Seed GI cestodes Goat Akhtar, 1988 Senecio lyratiparitus Leaves Anthelmintic Not reported Minja, 1989 Solanum nodiflorum Fruit Worm infestation Not reported Nwude and Ibrahim, 1980 Spigelia anthelmia Aerial parts H. contortus In vitro Assis et al., 2003 Linn. Swertia chirata Whole plants As. galli Not reported Shilaskar and Parashar, 1989 Tamarindus indica Root Round worms Cattle, goat, sheep ITDG and IIRR, 1996 Terminalia Leaf, root N. brasiliensis Rat Ibrahim et al., 1984 avicennoides Tiinospora rumphii Stem H. contortus Goat Fernandez, 1991 Tribulus terestris Plant As. galli Poultry Chakraborty et al., 1979

8 Table 1 Contd. Plant Part used Helminth (s)/Remarks Animal species Reference (s) Trichilia emetica Bark Fasciolosis, lungworms Cattle, goat, sheep, ITDG and IIRR, 1996 camel Uvaria hookeri Root-bark H. contortus Not reported Padmaja et al., 1993 Uvaria narum Vernonia Stem bark H. contortus In vitro Alawa et al., 2003 amygdalina Annona senegalensis Leaves Vernonia Seed GI nematodes Ruminants Nadkarni, 1954; Ikram and Hussain, anthelmintica 1978; Awan, 1981 Fruits/Seeds GI nematodes, cestodes Sheep, Goats Nadkarni, 1954; Chopra et al., 1956; Said, 1969; Awan, 1981; Singh et al., 1985; Shilaskar and Parashar, 1989; Javed and Oxyurids Not reported Akhtar, 1990 As. galli Chicken Oxyurids Mice Mehta and Parashar, 1966 Withania coagulans Not reported Earthworms In vitro Gaind and Budhiraja, 1967 Zanthoxylum alatum Essential oil Earthworms, roundworms Not reported Kokate and Varma, 1971; Mehta et al., 1981 Bark A. lumbricoides, F. buski, H. In vitro Singh et al., 1982 nana Not reported Earthworms, tapeworms, Not reported Kalyani et al., 1989 hookworms Zingiber officinale Rhizomes GI nematodes Sheep Iqbal et al., 2006c A. lumbricoides Human Kalesaraj, 1974, 1975 Anisakis larvae In vitro Goto et al., 1990 Dirofilaria immitis Canine Datta and Sukul, 1987; Chakraborty et al., 1994 S. mansoni Not reported Adewunmi et al., 1990

2.2. Plants used as antiprotozoals Protozoal diseases are always a threat to the animal and human health. Some plant extracts have been reported to possess antiprotozoal activity (Aguilar et al., 1994; Fournet et al., 1994; Atindehou et al., 2004; Hoet et al., 2004). For example, Cayaponia podantha Cogn., Helicteres gardneriana St., Melochia arenosa, Nectandra falcifolia and Paullinia elegans have been reported to possess antiprotozoal activities (Truti et al., 2005). Muelas-Serrano et al. (2000) reported the antitrichomonial effects of Chiranthodendron pentadactylon (fruit). Some of the commercialized antiprotozoal drugs developed from plants include: emetine from Cephaelis ipecacuhana, artemisinin from Artemisia annua and quinine from Cinchona species (Tagbota and Townson, 2001). Some of the plants having antiprotozoal effects are given in Table 2. Table 2: Plants used as antiprotozoals

Plant Part used Protozoa Test/Animal Reference (s) species Acacia nilotica Not reported Plasmodium (P.) falciparum In vitro Kirira et al., 2006 Acanthospermum hispidum Alkaloid P. falciparum In vitro Sanon et al., 2003 extracts Acronychia leavis Leaves Leishmania (L.) donovani In vitro Billo et al., 2005 Ajuga remota Not reported - P. falciparum In vitro Muregi et al., 2004 Albizia zygia Stem bark P. falciparum, Trypanosoma (T.) In vitro Ndjakou et al., 2007 brucei, T. rhodesiense and T. cruzi Alchornea cordifolia Leaves T. brucei brucei and P. falciparum In vitro Mustofa et al., 2000 Mesia et al., 2008 Alstonia macrophylla Not reported T. brucei brucei In vitro Camacho et al., 2003 Allanblackia monticola Fruit T. brucei rhodesiense In vitro Ndjakou et al., 2007 Amborella trichopoda Seeds T. cruzi In vitro Billo et al., 2005

9 Table 2 Contd. Plant Part used Protozoa Test/Animal Reference (s) species Annona muricata Not reported Leishmania spp. and T. cruzi In vitro Osorio et al., 2007

Annona purpurea Not reported T. brucei brucei In vitro Camacho et al., 2003 Artemisia afra Leaves Eimeria (E.) tenella, E. maxima and Broiler Naidoo et al., 2008 E. avervulina chickens Artemisia annua Not reported P. falciparum In vitro Sharma and Sharma, 1998 Aspidospernia megalocarpon Not reported P. falciparum, T. cruzi and In vitro Bernard et al., 2001 Leishmania spp. Azadirachta indica Not reported P. falciparum In vitro & in Omar et al., 2003; Kirira et vivo al., 2006 Babingtonia leratii Leaves L. donovani In vitro Billo et al., 2005 Bocconia frutescens Aerial parts Trichomonas (T.) vaginalis In vitro Calzada et al., 2007 Calophyllum caledonicum Bark L. donovani In vitro Billo et al., 2005 Calotropis procera Whole plant P. falciparum In vitro Sharma and Sharma, 2000 Campnosperma pana mense Not reported P. falciparum In vitro Bernard et al., 2001 Carica papaya Seeds T. vaginalis In vitro Calzada et al., 2007 Carissa edulis Not reported P. falciparum In vitro Kirira et al., 2006 Cayaponia podantha Not reported T. cruzi In vitro Truiti et al., 2005

Cerberiopsis candelabra Leaves and L. donovani In vitro Billo et al., 2005 Fruits Clerodendrum myricoides Not reported P. falciparum In vitro Muregi et al., 2004 Cocos nucifera Husk fibre T. vaginalis In vitro Calzada et al., 2007 Codiaeum peltatum Bark L. donovani In vitro Billo et al., 2005 Cola caricaefolia Not reported P. falciparum In vitro Menan et al., 2006 Combretum hartmannianum Leaves P. falciparum In vitro Ali et al., 2002 Combretum woodii Leaves E. tenella, E. maxima and E. Broiler Naidoo et al., 2008 avervulina chickens Conobea scoparioides Not reported P. falciparum T. cruzi and In vitro Bernard et al., 2001 Leishmania spp. Crossostylis multiflora Leaves and L. donovani In vitro Billo et al., 2005 Bark Cupaniopsis glomeriflora Leaves L. donovani In vitro Billo et al., 2005 Cucurbita maxima Fruit Histomonas (H.) meleagridis In vitro Grabensteiner et al., 2008 Cypholopus decipiens Leaves L. donovani In vitro Billo et al., 2005 Chrozophora senegalensis Stem and P. falciparum In vitro Benoit-Vical et al., 2008 Leaves Desmopsis panamensis Plasmodium spp. In vitro Osorio et al., 2007 Drosera neocaledonica Whole plant L. donovani In vitro Billo et al., 2005 Ekebergia capensis Not reported P. falciparum In vitro Muregi et al., 2004 Erythrina variegata var. Fastigiata Bark L. donovani In vitro Billo et al., 2005 Fagaropsis angolensis Not reported P. falciparum In vitro Kirira et al., 2006 Fagraea berteriana Leaves , Bark L. donovani In vitro Billo et al., 2005 and Flowers Flueggea virosa Leaves and P. falciparum In vitro Kaou et al., 2008 Roots Fontainea pancheri Leaves L. donovani In vitro Billo et al., 2005 Garcinia pedicillata Leaves L. amazonensis In vitro Billo et al., 2005 Gardenia urvillei Flowers L. donovani In vitro Billo et al., 2005 Geranium mexicanum Roots T. vaginalis In vitro Calzada et al., 2005 Glochidion billardieri Leaves and L. donovani In vitro Billo et al., 2005 Bark Guarea guidonia Not reported P. falciparum T. cruzi and In vitro Bernard et al., 2001 Leishmania spp. Guarea polymera Not reported P. falciparum T. cruzi and In vitro Bernard et al., 2001 Leishmania spp. Guatteria amplifolia Not reported P. falciparum, T. cruzi and In vitro Bernard et al., 2001 Leishmania spp. Harrisonia abyssinica Not reported P. falciparum In vitro Kirira et al., 2006 Harungana madagascarensis Seeds L. donovani In vitro Ndjakou et al., 2007 Holarrhena Africana Leaves T. brucei brucei In vivo mice Nwodoet al., 2007 Huberodendron patinoi Not reported P. falciparum T. cruzi and In vitro Bernard et al., 2001 Leishmania spp. Hygrophila guianensis Not reported P. falciparum T. cruzi and In vitro Bernard et al., 2001 Leishmania spp. Jacaranda caucana Not reported P. falciparum T. cruzi and In vitro Bernard et al., 2001 Leishmania spp.

10 Table 2 Contd. Plant Part used Protozoa Test/Animal Reference (s) species Lygodium venustum Aerial parts T. vaginalis In vitro Calzada et al., 2007 Marila laxiflora Not reported P. falciparum, T. cruzi and In vitro Bernard et al., 2001 Leishmania spp. Melochia arenosa Not reported T. cruzi In vitro Truiti et al., 2005 Melodinus scandens Leaves L. donovani In vitro Billo et al., 2005 Momordica charantia Whole plant T. brucei brucei In vitro Mesia et al., 2008 Murraya crenulata Wood L. donovani In vitro Billo et al., 2005 Myoporum crassifolium Wood L. donovani In vitro Billo et al., 2005 Myrica salicifolia Not reported P. falciparum In vitro Kirira et al., 2006 Myristica fatua Fruit L. donovani In vitro Billo et al., 2005 Nectandra falcifolia Not reported L. (Viannia) brasiliensis In vitro Truiti et al., 2005 Neoboutonia macrocalyx Not reported P. falciparum In vitro Kirira et al., 2006 Omphalocarpum glomerata Root Bark T. brucei brucei In vitro Mesia et al., 2008 Otoba novogranatensis Not reported P. falciparum T. cruzi and In vitro Bernard et al., 2001 Leishmania spp. Otoba parviflora Not reported P. falciparum T. cruzi and In vitro Bernard et al., 2001 Leishmania spp. Pagiantha cerifera Fruit L. donovani and L. amazonensis In vitro Billo et al., 2005 Pamianthe peruviana Not reported P. falciparum ,T. Brucei, T. In vitro Ali et al., 2002 rhodesiense and T. cruzi Pavetta crassipes Alkaloid extracts P. falciparum In vitro Sanon et al., 2003 Phyllanthus piscatorum Not reported P. falciparum and T. brucei In vitro Gertsch et al., 2004 Piper methysticum Leaves and Bark L. donovani In vitro Billo et al., 2005 Piptadenia africanum Stem bark T. cruzi, T. brucei brucei In vitro Mesia et al., 2008 Polyathia swaveleons Stem bark P. falciparum In vitro Mesia et al., 2008 Polygonum subsessile Leaves and Bark L. donovani In vitro Billo et al., 2005 Protium amplium Not reported P. falciparum T. cruzi and In vitro Bernard et al., 2001 Leishmania spp. Pseudomalmea boyacana Not reported Plasmodium In vitro Osorio et al., 2007 Rollinia exsucca Not reported Leishmania spp. ,T. cruzi and In vitro Osorio et al., 2007 Plasmodium Rollinia pittieri Leaves Leishmania spp. and T. cruzi In vitro Osorio et al., 2007 Rubus coriifolius Not reported Entamoeba (E.) histolytica and In vitro Alma et al., 2003 Giardia (G.) lambia. Salvadora persica Leaves P. falciparum In vitro Ali et al., 2002 Santalum austrocaledonicum Leaves and Bark L. donovani In vitro Billo et al., 2005 Sapium cornutum Stem bark P. falciparum In vitro Mesia et al., 2008 Scleria cf. polycarpa Aerial parts L. donovani In vitro Billo et al., 2005 Smilax orbiculata Leaves L. donovani In vitro Billo et al., 2005 Stephania abyssinica Not reported P. falciparum In vitro Muregi et al., 2004 Stereospermum Stem bark T. brucei rhodesiense In vitro Ndjakou et al., 2007 Strychnos heningsii Not reported P. falciparum In vitro Kirira et al., 2006 Swinglea glutinosa Not reported P. falciparum T. Cruzi and In vitro Bernard et al., 2001 Leishmania spp. Symphonia globulifera Leaf P. falciparum In vitro Ndjakou et al., 2007 Tabernaemontana obliqua Not reported P. falciparum T. cruzi and In vitro Bernard et al., 2001 Leishmania spp. Terminalia glaucescens Not reported P. falciparum In vitro Mustofa et al., 2000 Thymus vulgaris Not reported H. meleagridis In vitro Grabensteiner et al., 2008 Triclisia giletii Stem bark P. falciparum In vitro Mesia et al., 2008 Triclisia patens Not reported L. donovani In vitro Camacho et al., 2003 Tristaniopsis callobuxus Leaves, Bark and L. donovani In vitro Billo et al., 2005 Root Tristaniopsis glauca Leaves Not reported L. donovani In vitro Billo et al., 2005 Bark Tulbaghia violacea Whole plant E. tenella, E. maxima and E. Broiler Naidoo et al., 2008 avervulina chickens Uvaria afzelii Not reported P. falciparum In vitro Ménan et al., 2006 Vernonia colorata Aerial part P. falciparum In vitro Kaou et al., 2008 Vitis vinifera Seed E. tenella , E. maxima, E. Broiler Naidoo et al., 2008; avervulina and H. meleagridis chickens and Grabensteiner et al., 2008 In vitro Withania somnifera Not reported P. falciparum In vitro Kirira et al., 2006 Xylopia aromatic Not reported Leishmania spp. and T. cruzi In vitro Osorio et al., 2007 Zanthoxylum liebmannianum Leaves E. histolytica and G. lamblia In vitro Arrieta et al., 2001 Zanthoxylum tsihanimposa Stem bark P. falciparum In vitro Randrianarivelojosia et al., 2003 Zanthoxylum usambarense Not reported P. falciparum In vitro Kirira et al., 2006 Zieridium melicopaefolium Bark L. donovani In vitro Billo et al., 2005

11 2.3. Plants used as insecticides The insecticidal property of the plants evolved with the evolution of plants for over 400 million years ago. The plant kingdom has an enormous store of chemical substances used as defenses against pathogenic insects and microorganisms (Jacobson, 1983). In many parts of the world, various plant extracts and their different parts have been used for killing or repelling insects (Secoy and Smith, 1983). Approximately, more than 2400 plant species with pesticidal properties have been reported till 1988 (Grainge and Ahmed, 1988). Ethno-botanical insecticides are comparatively harmless and degradable in nature. They are cheap and easily available sources of biopesticides. The most famous phytochemical insecticides, Azadirachtin, and many other related compounds have been derived from Azadirachta indica. It has been extensively investigated in the fields of entomology and phytochemistry. Azadirachta indica contains many compounds with high concentration of Azadirachtin, the main insecticidal component, in seeds and leaves. These phytochemicals affect the insects/pests by their antifeedant, oviposition deterrent, fecundity suppressant and growth regulating activities (Mulla and Sut, 1999). Some of the plants with insecticidal properties are given in Table 3. Table 3: Plants used as insecticides

Plant Part used Insects/Remarks Animal Reference (s) species Achillea millefolium Not reported Mosquitoes Not reported Thomas et al., 2006 Alstonia boonei Leaf and Stem bark Sesamia calamistis Not reported Nathaniel et al., 2007 Apium graveolens Oil Aedes (Ae.) aegypti In vitro Chaiyasit et al., 2006 Achyrocline satureoides leaves and flowers Insecticide In vitro Arias et al., 1995 Artemisia absinthium Not reported Insecticide Not reported Jaenson et al., 2005 Linnaeus Azadirachta excels Not reported Mosquitoes Not reported Mulla and Sut, 1999 Azadirachta indica Leaves and Seeds Mosquitoes, flies, Not reported Mulla and Sut, 1999 Triatomines, Cockroaches, Fleas and Lice Seeds Damalinia limbata Goat Habluetzel et al., 2007 Azadirachta siamens Not reported Mosquitoes Not reported Mulla and Sut, 1999

Calpurnea aurea Not reported Lice In vitro Waka et al., 2004 Carum carvi Oil Ae. Aegypti In vitro Chaiyasit et al., 2006 Cassia sophera Not reported Rhyzopertha (Rh.) Not reported Belmain et al., 2001 dominica, Callosobruchus (Ca.) maculatus, Sitophilus (Si.) zeamais and Prostephanus (Pr.) truncates Chamaecrista nigrican Not reported Rh. dominica, Ca. Not reported Belmain et al., 2001 maculatus, Si. zeamais and Pr. Truncates Conyza newii Oil An. gambiae In vitro Omolo et al., 2005 Corymbia citriodora Leaves An. gambiae sensu lato, In vitro Seyoum et al., 2003 An. arabiensis and An. gambiae sensu strict Croton erythrochilus Not reported Brine shrimp In vitro Mongelli et al., 1995 Curcuma zedoaria Oil Ae. Aegypti In vitro Chaiyasit et al., 2006 Eucalyptus globules Not reported Mosquitoes In vitro Kweka et al., 2008 Ficus inspidia Not reported Brine shrimp In vitro Mongelli et al., 1995 Guarea kunthiana Stem Triatomine bug Not reported Coelho et al., 2006

12 Table 3 Contd. Plant Part used Insects/Remarks Animal Reference (s) species Guarea guidonia Root Triatomine bug Not reported Coelho et al., 2006 Helianthus annuus Not reported Pesticide Not reported De Cupere et al., 2001 Hyptis suaveolens Not reported Mosquitoes Not reported Thomas et al., 2006 Illicium verum Oil Ae. aegypti In vitro Chaiyasit et al., 2006 Lantana camara Not reported Mosquitoes In vitro Seyoum et al., 2002; Kweka et al., 2008; Seyoum et al., 2003 Leucas aspera Not reported An. subpictus and Cu. In vitro Kamaraj et al., 2008 Tritaeniorhynchus Lippia ukambensis Not reported An. gambiae sensu lato, In vitro Seyoum et al., 2003 An. arabiensis and An. gambiae sensu stricto Melia azedarach Not reported Mosquitoes Not reported Mulla and Sut, 1999 Melia toosendan Not reported Mosquitoes Not reported Mulla and Sut, 1999 Melia volkensii Not reported Mosquitoes Not reported Mulla and Sut, 1999 Mitragyna inermis Not reported Rh. dominica, Ca. Not reported Belmain et al., 2001 maculatus, Si. zeamais and Pr. truncatus Momordica charantia leaves Plutella xylostella Invitro Ling et al., 2008 Musa spp. Not reported Pesticide Not reported De Cupereet al., 2001 Myrica gale Not reported Insecticidal Not reported Jaenson et al., 2005 Not reported Mosquitoes Not reported Thomas et al., 2006 Neorautanenia mitis Not reported Lice Not reported Waka et al., 2004 Nicotiana glauca Not reported Mosquitoes In vitro Waka et al., 2004 Nicotiana tabacum Not reported Pesticide In vitro De Cupereet al., 2001; Seyoum et al., 2002 Ocimum americanum Not reported Mosquitoes In vitro Seyoum et al., 2002; Seyoum et al., 2003 Not reported Rh. dominica, Ca. Not reported Belmain et al., 2001 maculatus, Si. zeamais and Pr. truncatus Ocimum forskolei Shoots and leaves Mosquitoes Not reported Waka et al., 2004 Ocimum kilimandscharicum Oil and leaves Mosquitoes In vitro Kweka et al., 2008; Seyoum et al., 2003 Ocimum suave Oil and seeds Mosquitoes In vitro Kweka et al., 2008; Seyoum et al., 2003 Origanum acutidens Aerial parts Si. granarius and Tribolium Not reported Kordali et al., 2008 confusum Origanum vulgare Not reported Bemisia tabaci Not reported Calmasur et al., 2006 Otostegia integrifolia Not reported Fleas Not reported Waka et al., 2004 Piper longum Oil Ae. aegypti In vitro Chaiyasit et al., 2006 Piper gaudichaudianum Viridiflorol, Ae. aegypti In vitro Morais et al., 2007 aromadendrene and β- selinene Piper humaytanum β,-selinene and Ae. aegypti In vitro Morais et al., 2007 Caryophyllene oxide Piper permucronatum Dillapiol and Myristicin Ae. aegypti In vitro Morais et al., 2007 Piper hostmanianum Saricin and Myristicin Ae. aegypti In vitro Morais et al., 2007 Plectranthus marruboides Oil An. gambiae In vitro Omolo et al., 2005 Quercus infectoria Not reported An. stephensi In vitro Aivazi and Vijayan, 2009 Rhinacanthus nasutus Not reported An. subpictus and Cu. Not reported Kamaraj et al., 2008 tritaeniorhynchus Rhododendron tomentosum Not reported Insecticidal Not reported Jaenson et al., 2005 Salvia shimperi Not reported Fleas Not reported Waka et al., 2004 Sapium marmieri Not reported Brine shrimp In vitro Mongelli et al., 1995 Securidaca longepedunculata Not reported Rh. dominica, Ca. Not reported Belmain et al., 2001 maculatus, Si. zeamais and Pr. truncatus Simarouba glauca Not reported Pesticide Not reported De Cupere et al., 2001

Simarouba versicolor Root bark Triatomine bug Not reported Coelho et al., 2006 Solanum torvum Not reported An. subpictus and Cu. Not reported Kamaraj et al., 2008 tritaeniorhynchus Synedrella nodiflora Not reported Rh. dominica, Ca. Not reported Belmain et al., 2001 maculatus, Si. zeamais and Pr. truncatus Tagetes minuta Not reported Mosquitoes In vitro Seyoum et al., 2002 Talauma ovate Stem wood Triatomine bug Not reported Coelho et al., 2006

13 Table 3 Contd. Plant Part used Insects/Remarks Animal Reference (s) species Thujopsis dolabrata var. Not reported Reticulitermes speratus, Not reported Ahn et al., 2004 hondai Lasioderma serricorne, Ca. chinensis, Si. oryzae, Plutella xylostella, Myzus persicae, Blatella germanica, and Tetranychus urticae Uvaria acuminate Not reported brine shrimp In vitro Massele and Nshimo, 1995 Verbascum cheiranthifolium Flowers Rh. dominica Not reported Khoshnoud et al., 2008 Vitex negundo Not reported An. subpictus and Cu. Not reported Kamaraj et al., 2008 tritaeniorhynchus

2.4. Plants used as acaricides The role of plants in the control of arachnids (Tabassam et al., 2008) has gained interest due to the development of resistance against synthetic preparations and serious health hazards for consumers due to their residual and environmental effects (Schnitzerling et al., 1989; Mendes et al., 2001; Weigel et al., 2002; Aquino et al., 2004). Use of plants or oils extracted from these plants as acaricidals is considered safe, environment friendly, efficient and convenient (Koul and Dhaliwal, 2001). Use of plants as acaricides and repellants has been considered as a traditional remedy (Aligiannis et al., 2001; Çalmaşuret al., 2006). Azadirachta indica (Benavides et al., 2001), Gynandropsis gynandra (Lwande et al., 1999), Cleome hitra (Ndungu et al., 1999) and Tamarindus indica (Chungsamarnyart and Jansawan, 2001) have been reported for their acaricidal activity. Some of the plants possessing acaricidal activity are given in Table 4. Table 4: Plants used as acaricides

Plant Part used Parasite Animal Reference (s) species Acanthus ebracteatus Not reported Rhipicephalus (R.) microplus In vitro Chungsamarnyart et al., 1988 Achillea millefolium Not reported Ixodes (I.) ricinus Not reported Thorsell et al., 2006 Acorus calamus Rhizome R. microplus In vitro Chungsamarnyart et al., 1988; Pathak et al., 2004 Ageratum houstonianum Leaf R. lunulatus Not reported Pamo et al., 2005 Allium sativum Bulb Hyalomma (Hy.) marginatum In vitro Nchu et al., 2005 rufipes Not reported R. pulchellus Not reported Nchu et al., 2005 Oil Sarcoptes (S.) scabiei var. suis Not reported Magi et al., 2006 Annona squamosa Not reported R. microplus Not reported Chungsamarnyart et al., 1988 Artemisia abrotanum Not reported I. ricinus Not reported Tunon et al., 2006 Artemisia absinthium Not reported I. ricinus Not reported Jaenson et al., 2005 Not reported S. scabiei var. suis Not reported Magi et al., 2006 Not reported Two spotted spider mite In vitro Chiasson et al., 2001 Artemisia vulgaris Not reported S. scabiei var. suis Not reported Magi et al., 2006 Azadirachta Indica Oil R. microplus Not reported Thakur et al., 2007 Bark R. microplus Not reported Pathak et al., 2004; Maharaj et al., 2005 Leave Tick Not reported Pathak et al., 2004 Seed Hy. dromedarii Not reported Al-Rajhy et al., 2003 Not reported R. pulchellus Not reported Handule et al., 2002 Not reported Hy. Anatolicum excavatum Not reported Abdel-Shafy and Zayed, 2002 Brassica juncea Not reported Dermanyssus (Dy.) gallinae In vitro Kim et al., 2004 Cananga odorata Oil Dermatophgoides (De.) farinae In vitro Rim and Jee, 2006 and D. pteronyssinus

14 Table 4 Contd.. Plant Part used Parasite Animal Reference (s) species Callicarpa americana Not reported I. scapularis, Not reported Carroll et al., 2007 Amblyomma (A.) americanum Calocedrus decurrens Wood I. scapularis Not reported Dolan et al., 2007 essential oil Chamaecyparis lawsoniana Wood I. scapularis Not reported Dolan et al., 2007 essential oil Citrus Spp. Peel oil R. microplus Not reported Chungsamarnyart and Jansawan, 1996 Cleome hirta Essential oil R. appendiculatus Not reported Ndungu et al., 1999 Cloves Not reported I. ricinus Not reported Thorsell et al., 2006 Commiphora molmol Not reported Argas persicus Not reported Massoud et al., 2005 Copaifera reticulate Oleoresin R. microplus Not reported Fernandes and Freitas, 2007 Coriandrum sativum Not reported Dy. gallinae In vitro Kim et al., 2004 Cochlearia armoracia Not reported Dy. gallinae In vitro Kim et al., 2004 Cymbopogon citratus Oil De. farinae and D. In vitro Rim and Jee, 2006 pteronyssinus. Cymbopogon winterianus Essential oil R. microplus Not reported Martins, 2006 Cymbopogon nardus Oil S. scabiei var. suis Not reported Magi et al., 2006 Cymbopogon winterianus Oil Tyrophagus (Ty.) putrescentiae In vitro Kim et al., 2003 Cymbopogon citratus Oil Ty. putrescentiae In vitro Kim et al., 2003 Dahlstedtia pentaphylla Root R. microplus Not reported Pereira and Famadas, 2004 Dianthus caryophyllum Flower I. ricinus Not reported Tunon et al., 2006 Drimys brasiliensis Essential oil R. microplus, R. sanguineus Not reported Ribeiro et al., 2008 of stem and leaf Eucalyptus Oil R. microplus Not reported Thakur et al., 2007 Not reported I. ricinus Not reported Thorsell et al., 2006 Eucalyptus globulus Oil S. scabiei var. suis Not reported Magi et al., 2006 Gynandropsis gynandra Essential oil R. appendiculatus Not reported Malonza, 1992; Lwande et al., 1999 Not reported A. variegatum Not reported Malonza, 1992 Heracleum sosnowskyi Not reported S. scabiei var. suis Not reported Magi et al., 2006 Hypericum polyanthemum Not reported R. microplus Not reported Ribeiro et al., 2007 Juniperus occidentalis Wood I. scapularis Not reported Dolan et al., 2007 essential oil Juniperus communis Oil S. scabiei var. suis Not reported Magi et al., 2006 Juniperus oxycedrus Not reported Dy. gallinae In vitro Kim et al., 2004 Lavandula officinalis Not reported R. annulatus Not reported Abdel-Shafy and Soliman, 2004 Laurus nobilis Oil Ty. putrescentiae In vitro Kim et al., 2003 Ledum palustre Not reported I. ricinus Not reported Jaenson et al., 2005 Luffa acutangula Not reported R. microplus Not reported Chungsamarnyart et al., 1988 Margaritaria discoidea Not reported R. appendiculatus, A. Not reported Kaaya et al., 1995 variegatum Marjorana hortensis Not reported R. annulatus Not reported Abdel-Shafy and Soliman, 2004 Matricaria chamomile Flower R. annulatus Not reported Pirali-Kheirabadi and Razzaghi- Abyaneh, 2007 Melaleuca alternifolia Not reported I. ricinus Not reported Iori et al., 2005 Melaleuca alternifolia Oil S. scabiei var. suis Not reported Magi et al., 2006 Melia azedarach Leaf R. microplus Not reported Matias et al., 2003 Fruit R. microplus Not reported Borges et al., 2003 Melinis minutiflora Not reported R. microplus Not reported Prates et al., 1993; Muro et al., 2004 Mentha piperita Not reported R. annulatus Not reported Abdel-Shafy and Soliman, 2004 Mentha pulegium Oil S. scabiei var. suis Not reported Magi et al., 2006 Mentha pulegium Oil De. farinae and D. In vitro Rim and Jee, 2006 pteronyssinus Mentha viridis Not reported R. annulatus Not reported Abdel-Shafy and Soliman, 2004 Myrica gale Not reported I. ricinus Not reported Jaenson et al., 2005 Myristica fragrans Oil Ty. putrescentiae In vitro Kim et al., 2003 Nicotiana tabacum Leaf R. haemaphysaloides Not reported Choudhary et al., 2004 Ocimum basilicum Not reported R. annulatus Not reported Abdel-Shafy and Soliman, 2004 Ocimum suave Leaf R. appendiculatus Not reported Mwangi et al., 1995 Origanum vulgare Oil Ty. putrescentiae In vitro Kim et al., 2003 Pimenta dioica Bark and leaf R. microplus Not reported Brown et al., 1998 Oil Ty. putrescentiae In vitro Kim et al., 2003 Piper nigrum Oil S. scabiei var. suis Not reported Magi et al., 2006 Psiadia punctulata Aerial parts R. appendiculatus and In vitro Nanyingi et al., 2008 Boophilus decoloratus Pongamia pinnata Oil R. microplus Not reported Thakur et al., 2007 Rhododendron tomentosum Not reported I. ricinus Not reported Jaenson et al., 2005

15 Table 4 Contd. Plant Part used Parasite Animal Reference (s) species Sapindus saponaria Stem peal R. microplus Not reported Fernandes et al., 2005 Stemona collinsae Not reported R. microplus Not reported Chungsamarnyart et al., 1988 Stylosanthes hamata Not reported R. microplus Not reported Muro et al., 2003 Stylosanthes humilis Not reported R. microplus Not reported Muro et al., 2003 Syzgium aromaticum Bud or leaf oil Ty. putrescentiae In vitro Kim et al., 2003 Tamarindus indicus Not reported R. microplus Not reported Chungsamarnyart and Jansawan, 2001 Tanacetum vulgare Not reported S. scabiei var. suis Not reported Magi et al., 2006 Not reported Two spotted spider mite In vitro Chiasson et al., 2001 Thymus zygis (Thyme white) Oil Ty. putrescentiae In vitro Kim et al., 2003 Thymus vulgaris (Thyme red) Oil Ty. putrescentiae In vitro Kim et al., 2003

Vitex agnus Not reported I. ricinus, R. sanguineus Not reported Pathak et al., 2004; Mehlhorn et al., 2005

2.4. Scientific validation Scientific validation of folk believes not only helps to understand the technology of the consumers but, it also helps to satisfy the stakeholders for adaptation of local technology by providing scientific data on activity. The foresightedness of the research managers lies in bringing the best of science and healing together (Dwivedi, 1998). Existing local technologies need to be included in an improved package of animal health care and production after their scientific validation (Yadav, 2007). A strong need of integrating the allopathic and EVM for sustainable advance can be fulfilled by knowing the active ingredients present in effective plants. Table 5 presents methods used by various workers to evaluate the anthelmintic, antiprotozoal and acaricidal activities of plants. Table 5: Tests/assays for scientific evaluation of ethnobotanicals

Test In vivo/ in References vitro Anthelmintic activity Adult motility assay In vitro Iqbal et al., 2007; Jabbar et al., 2007; Eguale et al., 2006; Hounzangbe- Adote et al., 2005; Paolini et al., 2003 Egg hatch test In vitro Iqbal et al., 2007; Jabbar et al., 2007; Eugale et al., 2006; Hounzangbe- Adote et al., 2005; Molan et al., 2003; Ketzis et al., 2002; Pessoa et al., 2002; Mahajan et al., 1985; Molan et al., 2002; Niezen et al., 2002 Larval development assay In vitro Ademola et al., 2005; Ademola et al., 2004; Molan et al., 2003; Niezen et al., 2002; Molan et al., 2002; Athanasiadou et al., 2001 Fecal egg count reduction test In vivo Iqbal et al., 2007; Jabbar et al., 2007; Eugale et al., 2006; Ademola et al., 2005; Ademola et al., 2004; Githiori et al., 2004; Hordegen et al., 2003; Ketzis et al., 2002; Onyeyili et al., 2001; Kailani et al., 1995; Akhtar and Aslam, 1989; Akhtar and Riffat, 1986; Riffat et al., 1986; Akhtar and Javed, 1985; Niezen et al., 1998; Niezen et al., 1998a; Butter et al., 2000; Athanasiadou et al., 2001a; Heckendorn et al., 2007; Min et al., 2004; Niezen et al., 2002; Kahiya et al., 2003; Paolini et al., 2003; Shaik et al., 2004; Paolini et al., 2005 Larval migration inhibition assay In vitro Barrau et al., 2005; Hounzangbe-Adote et al., 2005; Molan et al., 2003; Paolini et al., 2003; Molan et al., 2004; Paolini et al., 2004; Molan et al., 2003a; Molan et al., 2000; Lorimer et al., 1996 Antiprotozoal activity Radioactive micromethod In vitro Menan et al., 2006 In vitro semi-automated micro-dilution assay In vitro Desjardins et al., 1979; Muregi et al., 2003 technique Lactate deshydrogenase assay In vitro Mesia et al., 2008 In vitro susceptibility assays In vitro Cedillo-Rivera et al., 2002; Calzada et al., 2007 Antitrypanosomal activity and cytotoxicity In vitro Baltz et al., 1985; Raz et al., 1997 Microculture radioisotope technique In vitro Desjardins et al., 1979; Ridley et al., 1996

16 Table 5 Contd. Test In vivo/ in References vitro Antileishmanial assay In vitro Camacho et al., 2002 Antitrypanosomal assay In vitro Billo et al., 2005 ; Camacho et al., 2002 Cytotoxicity assay In vitro Anderson et al., 1991 Antileishmanicidal assay In vitro M’Bongo et al., 1997; Okpekon et al., 2004 Sister chromatid exchange assay In vitro Arias et al., 1995 Artemia salina test In vitro Arias et al., 1995 Acaricidal activity Direct contact application method In vitro Kim et al., 2004; Lee et al., 2006; Rim and Jee, 2006 Fumigation method. In vitro Kim et al., 2004; Rim and Jee, 2006 Filter paper contact bioassay, In vitro Kim et al., 2004 Impregnated fabric disc bioassay In vitro Kim et al., 2003 Ovicidal Activity In vitro Chiasson et al., 2004 Contact Efficacy against Adult Stage. In vitro Chiasson et al., 2004 Larval immersion test In vitro Sabatini et al., 2001 Modified Larval packet test In vitro Al-Rajhy et al., 2003 Insecticidal activity Adult parasitoid feeding experiments In vitro Ruiu et al., 2008 Tunnel chamber experiment In vitro Kweka et al., 2008 Cage tests In vitro Kweka et al., 2008 Susceptibility bioassays In vitro Kweka et al., 2008; Chaiyasit et al., 2006 Contact or cone bioassays In vitro Kweka et al., 2008 Antifeedant activity In vitro Ling et al., 2008 Immature house fly feeding experiments In vitro Ruiu et al., 2008 Thermal expulsion method In vitro Seyoum et al., 2002; Seyoumet al., 2003 Topical test In vitro Coelho et al., 2006; Chaiyasit et al., 2006 Direct burning method In vitro Seyoum et al., 2002 Fumigant toxicity In vitro Omolo et al., 2005 Brine shrimp toxicity microplate bioassay In vitro Mongelli et al., 1995; Massele and Nshimo, 1995 Larvicidal bioassay In vitro Aivazi and Vijayan, 2009; Morais et al., 2007 Moulting inhibition In vitro Arias et al., 1995 Sub-lethal bioassay In vitro Ruiu et al., 2008

Conclusion Based on the above review, it is concluded that (i) the indigenous ways of treatment emerging from the traditional medicinal practices are great assets of different communities, and (ii) plants have great potential to be used in their crude forms or as extracts in the light of empirical evidence for their efficacy as antiparasitics. Therefore, there is strong need to (i) document the indigenous knowledge in traditional medical/veterinary medicinal systems, (ii) validate the EVM using standard scientific procedures, and (iii) carry out researches on bioactive plants for drug discovery.

17 Chapter # 3 Materials and Methods The objectives of the present study were to (i) document the ethno-veterinary knowledge with particular reference to the antiparasitic practices in some selected parts of District Jhang (Pakistan), and (ii) to validate the claims of local healers regarding anthelmintic activity of some plants in animals. The following procedures were adopted to achieve the objectives of the study. 3.1. Documentation of EVM with Particular Reference to the Anti-parasitic Practices 3.1.1. Study area (Fig. 1: Map of Pakistan showing the study area) Jhang, formerly known as Jhangi Sial, is one of the historically oldest districts of subcontinent, which was established around 2000 BC. District Jhang lies between 30o-37o to 31o-59o north latitudes and 71o-37o to 73o-13o east longitudes. It is located in central Punjab of Pakistan and is bordered by the districts Sargodha in north, Gujranwala in northeast, Faisalabad and Toba Tek Singh in east, Layya and Bhakkar in west and Khushab in northwest and in south by Khanewal and Muzaffargarh. It is divided into four Tehsils (administrative subdivisions of district) that is, Chiniot, Shorkot, Jhang and Ahmad Pur Sial. The climate is extreme hot and cold throughout its area. The total area of this district is 8,809 km2 having two rivers Chenab and Jhelum and a large area of the district consists of sand dunes and barren land. The study area in present study was located at eastern bank of River Chenab in the premises of Thesil Chiniot, Jhang and Shorkot. This is a riverside belt about 4 x 70 km in size spreading from Chiniot to Shorkot. The following villages/small towns of district Jhang were included in the study: Tehsil Shorkot 1. Dab Kalan, 2. Binda Surbana, 3. Budh Rajbana Shumail, 4. Hassu Wal, 5. Allah Yar Joota, 6. Kakoowala, 7. Qaim Bharwana, 8. Budhuwana, 9. Bela Surbana, 10. Haveli Bahdur, 11. Shah Gharbi Tehsil Chiniot 1. Thatta Muhammad Shah, 2. Bhawana, 3. Maingni, 4. Naushehra, 5. Kurk Muhammandi, 6. Bukhari, 7. Mathrumma, 8. Kazian, 9. Harse Sheikh, 10. Jehangiar Galotiran Tehsil Jhang 1. Kot Rustam, 2. Kot Dewan, 3. Dheedoana, 4. Pirkot Sadhana, 5. Jharki, 6. Patoana, 7. Thatta Khuriana, 8. Chund Bharwana, 9. Shah Jewana, 10. Jogera

18 Fig.1. Map of Pakistan showing the study area

19 3.1.2. Demographic Characteristics of the Study Area The human population of District Jhang is 2,834,545. The riverside belt is rich in livestock due to its geographical location and availability of lush green pastures. Livestock farming is very important for the local population in the study area, as traditionally social status of a family is assessed by the number of animals, especially cattle and buffaloes, owned by the family. Total livestock population in the district is 50,62,387 heads with 8,72,819 cattle, 11,75,170 buffaloes, 3,85,050 sheep, 10,06,992 goats, 8,289 camels, 15,123 horses, 1,310 mules, 1,72,397 asses and 14,25,237 poultry (Economic Survey of Pakistan, 2006). The women are encouraged to help their family in raising animals and actively participating in the agricultural operations. Therefore, various aspects of folk medical knowledge are held in common without the bias of gender. Different ethnic and regional groups, however, additionally hold specific folk knowledge. There is also occupational specialization, for example farmers know much about ruminant ethno-veterinary practices. 3.1.3. Collection of Information on EVM The population of interest was individuals knowledgeable on EVM, locally called as “Sianas” (experts). Only the male population was selected for documentation of EVM, as women were not allowed by their family elders for interviews for collection of information. The data on EVM was collected following the definition of veterinary anthropology (Mathias- Mundy and McCorckle 1989). Initially, an exploration survey was conducted with local veterinary officer and veterinary assistant to identify the traditional healers and farmers in the study area. This primary survey was in fact a Rapid Rural Appraisal (RRA), which was followed by actual interview with the respondents identified during RRA. A survey team was formed to conduct the interviews with respondents. Survey team included a veterinarian, veterinary assistant and community leader. The survey team was clearly informed to document information on remedies based on plants and other materials (if any) being used in animals. The interviews were conducted in the local language i.e., Punjabi. The data collection was divided into the following three parts. 1. Using the questionnaire method to generate a purposive sample of ethno-veterinary key respondents. A total of 250 key respondents were interviewed. Key respondents were the traditional healers or other people in the study area with a profound knowledge of a particular issue or technology (Waters-Bayer and Bayer, 1994). They had a more extensive vocabulary of local social and cultural systems than others in a community (Etkin, 2002). A purposive sample means identifying and interviewing that particular

20 subset of knowledgeable people. Intensive collaboration with key respondents was considered to be a particularly effective research strategy as emphasized by Etkin (2002). 2. Conducting interviews with key respondents, veterinarians, and the field staff of the public and/or private sector (s). 3. Holding focused group discussions with the key respondents. 4. Veterinarian in the survey team ensured that the nomenclature of the diseases, plants and other materials used by the traditional healers and farmers is correct scientifically. The suspicious information and that which lacked verification was discarded. All the respondents were asked to show the species of plants in the field and samples were collected for identification. Collected samples were identified by a botanist at the Department of Botany, University of Agriculture, Faisalabad-Pakistan (UAF) and voucher specimens were preserved at the Herbarium, Ethno veterinary Research and Development Center, Department of Parasitology, UAF. 3.2. Validation of Anthelmintic Activity 3.2.1. Collection of Plant Materials A total of 19 of the total 46 plants documented for their use in different ailments/conditions in animals were used as anthelmintic. Seven (Table 9) of these 19 plants/parts of plant were selected for evaluation of their anthelmintic activity. The criterion for the selection of plants for anthelmintic activity was their convenience of availability to the farmer. The criteria for selection of plants for anthelmintic activity were (i) cost effectiveness, (ii) ready availability and (iii) no or little scientific validation for deworming efficacy. These plants included Acacia nilotica (L.) Willd. ex Delile, Amomum subulatum Roxb., Vitex negundo L., Arundo donax L., Ferula assa-foetida L. and Areca catechu L. The selected plants were collected from field and/or purchased from the local market. Voucher specimens were kept at the Herbarium, Ethno veterinary Research and Development Center, Department of Parasitology, UAF, after authentication of plants from a botanist at the Department of Botany, UAF. 3.2.2. Extraction and Fractionation Extraction: The plant materials were dried under shade and ground into fine powder. Crude aqueous-methanol extracts (CAME) were prepared by following the methods of Tabassam et al. (2008). Briefly, powdered plant material was soaked in sufficient quantity of solvent (aqueous- methanol 30:70) for three days. After which, the filtrate was collected through a piece of muslin cloth and filter paper. This process of soaking and filtration of extract was repeated for a total of three times. The combined filtrate was evaporated in a rotary evaporator at 40 °C under reduced

21 pressure to yield the CAME and percent yield was recorded on dry matter basis using the following formula: Percentage yield (%) = (A-B/A) x 100 Where, A = weight of dry matter before dipping, and B = weight of dry matter after dipping Fractionation: fractionation of all the crude extracts was done using three different organic solvents, i.e., chloroform, petroleum spirit and ethyle acetate (Williamson et al., 1998). Briefly, 20 g of crude extract was dissolved in 20 ml distilled water and poured into a separating funnel. Then, for preparation of first fraction, 60 ml of petroleum spirit was added into the same separating funnel, already containing CAME, and funnel was shaken vigorously. This mixture of CAME solution and petroleum spirit, in separating funnel, was kept undisturbed for 30 min to let the two layers separate. The layer of petroleum spirit, containing the soluble part of extract, was separated and 60 ml of petroleum ether was added again. This procedure was repeated until a clear layer of petroleum spirit was obtained (indicating the complete removal of petroleum spirit soluble fraction from CAME). All the solvent (petroleum spirit) was combined and evaporated in a rotary evaporator to obtain the petroleum spirit fraction. Then, the extract was treated with chloroform in the same manner to obtain chloroform fraction. Remaining portion of extract was fractioned with ethyl acetate, adopting the procedure described previously. After separating three different fractions the remaining material was treated as the aqueous portion and concentrated to yield the aqueous fraction. Different fractions from CAME were weighed and recorded. Table 6: Plants selected for anthelmintic activity Sr. Plant species Plant family Part/s Vernacular No. used name 1 Acacia nilotica (L.) Willd. ex Delile Fabaceae Leaves Desi Kikar 2 Acacia nilotica (L.) Willd. ex Delile Fabaceae Bark Desi Kikar 3 Amomum subulatum Roxb. Zingiberaceae Seeds Bari Ilaichi 4 Vitex negundo L. Verbenaceae Seeds Smalu 5 Arundo donax L. Poaceae Leaves Nara 6 Ferula assa-foetida L. Umbelliferae Latex Heang 7 Areca catechu L. Arecaceae Seeds Supari

3.2.3. Parasitological Procedures 3.2.3.1. Parasites (Alawa et al., 2003) Adult H. contortus worms were obtained from the abomasal contents of slaughtered sheep. Some of the worms were kept separate to be used in adult motility assay (section 3.2.3.2); whereas, from the remaining worms, females were separated and crushed in mortar and pestle to liberate the eggs. These eggs were cultured in autoclaved sheep feces and vermiculite in a jar.

22 The jar was left standing at room temperature for one week. Larvae were obtained from fecal culture by rinsing the moisture droplets from the sides of the culture jar. Two worm free lambs were artificially infected with these L3 larvae @ 10,000 larvae per lamb to obtain the fresh eggs for subsequent in vitro egg hatch assays. 3.2.3.2. Adult Motility Assay Mature live Haemonchus (H.) contortus from sheep were used to determine the effect of crude extracts and different fractions by the method described previously by Singh et al. (1985). The mature worms were collected from the abomasums of freshly slaughtered sheep in the local abattoir. The worms were washed and finally, suspended in phosphate buffered saline (PBS). A minimum of 10 worms were exposed in three replicates to each of the following treatments in separate Petri dishes/test tubes at room temperature (25-300C). 1. Crude aqueous methanol extract @ 50, 25, 12.5, 6.25, 3.12 and 1.56 mg/ml 2. Petroleum spirit fraction @ 50, 25, 12.5, 6.25, 3.12 and 1.56 mg/ml 3. Chloroform fraction @ 50, 25, 12.5, 6.25, 3.12 and 1.56 mg/ml 4. Ethyl acetate fraction @ 50, 25, 12.5, 6.25, 3.12 and 1.56 mg/ml 5. Levamisole @ 0.55 mg/ml 6. PBS The inhibition of motility and/or mortality of worms kept in different treatments were used as criterion for anthelmintic activity. The motility was observed on 0, 2, 4, 6, 8, 10, 12 and 24 hr intervals. Finally, the treated worms were kept for five minutes in the lukewarm fresh PBS for the revival of motility. The number of dead and survived worms was recorded for each treatment. 3.2.3.3. Egg Hatch Assay The egg hatch assay was carried out using the World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P) guidelines for determining anthelmintic resistance (Coles et al., 1992) with modifications that allowed testing of the natural compounds (Alawa et al., 2003). Egg hatch assays comprised of the following steps: (i) Collection of eggs for assay For collection of eggs, fecal material was collected from infected sheep and washed with distilled water. Washed fecal pellets were mashed in distilled water to form a liquid suspension of fecal material. This fecal suspension was passed through a 100-mesh (150 µm pore size) sieve. The sieved suspension was again passed through another sieve of 400 meshes (38 µm pore size) and contents on sieve were washed with warm water. The material left on the sieve was transferred into 50 ml tubes (about 10 ml fecal material/tube) and tubes were filled with

23 water. These tubes were centrifuged at 1,000 rpm for 5 min, after removing the supernatant; sediments were transferred to another set of tubes. Five ml of MgSO4 (1.2 specific gravity) was added to each tube and contents were mixed gently. The tubes were then filled with the MgSO4 solution and centrifuged at 1500 rpm for 5 minutes, and supernatant was washed through a 400- mesh sieve. The retained eggs were transferred into a 50 ml tube. (ii) Test procedure The crude extract, and its petroleum spirit, chloroform, ethyl acetate and aqueous fractions were used as active treatments to study the anthelmintic activity. Albendazole, a commercially available anthelmintic, was used as a positive control. The tests were performed in 24-multiwell plates. Approximately, 250 eggs in 1.5 ml of water were placed in each well of the 24-multiwell plate. Five different concentrations (serial dilutions) of each plant extract/fraction (initially diluted in DMSO; maximal DMSO concentration was 0.5%) and albendazole were placed in each treatment well as follows: 1. Crude aqueous methanol extract: 12, 1.2, 0.12, 0.012 and 0.0012 mg/ml 2. Petroleum spirit fraction: 12, 1.2, 0.12, 0.012 and 0.0012 mg/ml 3. Chloroform fraction: 12, 1.2, 0.12, 0.012 and 0.0012 mg/ml 4. Ethyl acetate fraction: 12, 1.2, 0.12, 0.012 and 0.0012 mg/ml 5. Albendazole: 25, 2.5, 0.25, 0.025, and 0.0025 µg/ml All treatments were randomly allocated to the wells and experiment was repeated for three times. The plates were incubated at 28°C for 36 h, following which, percent of hatched larvae (alive or dead) and eggs were counted under an inverted microscope. 3.2.3.4. Fecal egg count reduction test (i) Study animals For each plant material, 48 (minimum) four to eight months old sheep, naturally infected with mixed species of gastrointestinal nematodes (GINs), were procured from different private livestock farms (Appendix 1) of Punjab-Pakistan. Before the start of the experiment, EPG was determined following standard parasitological procedures and McMaster technique (Urquhart et al., 2003). For nematode species composition, coproculture was performed for identification of larvae using standard description of MAFF (1986) which revealed a mixed infection of GINs including mainly (>80%) Haemonchus contortus. The other species were Teladorsagia circumcincta, Trichostronglyus spp. and Trichuris ovis. Animals with EPG exceeding than 750 eggs g-1 of feces were selected for the in vitro trials. The animals were washed with an effective insecticide. The sheep were kept on wood shaving and stall fed on a maintenance ration and water was offered ad libitum.

24 Appendix 1: List of private livestock farms included in the study Sr. No. Name of Farm Address 1. Talib Sheep Farm Chak No. 209R.B Faisalabad 2. Anwar Sheep farm Chak No. 248 R.B Faisalabad 3. Jehangir Sheep Farm Chak No. 71 G.B Faisalabad 4. Gujar Sheep Farm Chak No. 243R.B Faisalabad 5. Irfan Sheep Farm Chak No. 248 R.B Faisalabad 6. Lashari Sheep Farm Chak No. 243R.B Faisalabad 7. Ali Sheep Farm Chak No. 240R.B Faisalabad (ii) Treatment and follow-up procedures Prior to treatment, fecal samples were obtained directly from rectum of each animal, at least three times at interval of three days. On each occasion, the detailed morphological identification of the respective eggs was studied according to the defined characteristics of the genus as described by MAFF (1986). On day 0, sheep were divided into eight groups from 1 to 8 according to complete randomized design, taken into consideration their live weight and EPG. Layout plans for evaluating the in vivo anthelmintic activity of Acacia nilotica, Vitex negundo, Arundo donax, Amomum subulatum, Areca catechu and Ferula assa-foetida are given in Tables 7, 8 and 9. The doses of the plants were based on their traditional use by the farmers, and given once per os wrapped in a piece of newspaper. Table 7: Layout plan for crude powder and crude aqueous methanolic extract of Acacia nilotica (Bark & Leaves), Vitex negundo (seeds) and Arundo donax (leaves) given to different groups of sheep naturally infected with mixed species of gastrointestinal nematodes Groups Treatments 1 Untreated control 2 Levamisole HCl (Nilverm® 1.5%, w/v; ICI Pakistan Limited, Animal Health Division) at 7.5 mg kg−1 body weight (b.wt.) 3 Crude powder (CP) at 1 g kg−1 b.wt. 4 CP at 4 g kg−1 b.wt. 5 CP at 7 g kg−1 b.wt. 6 Crude aqueous methanolic extract (CAME) at equivalent dose rate 1 g kg−1 b.wt. of CP 7 CAME at the equivalent dose rate 4 g kg−1 b.wt. of CP 8 CAME at the equivalent dose rate 7 g kg−1 b.wt. of CP *CP = Crude powder; CAME = Crude aqueous methanol extract Table 8: Layout plan for crude powder and crude aqueous methanolic extract of Amomum subulatum (Fruit) given to different groups of sheep naturally infected with mixed species of gastrointestinal nematodes Groups Treatment 1 Untreated control 2 Levamisole HCl (Nilverm® 1.5%, w/v; ICI Pakistan Limited, Animal Health Division) at 7.5 mg kg−1 body weight (b.wt.) 3 Crude powder (CP) at 0.5 g kg−1 b.wt. 4 CP at 1 g kg−1 b.wt. 5 CP at 3 g kg−1 b.wt. 6 Crude aqueous methanolic extract (CAME) at equivalent dose rate 0.5 g kg−1 b.wt. of CP 7 CAME at the equivalent dose rate 1 g kg−1 b.wt. of CP 8 CAME at the equivalent dose rate 3 g kg−1 b.wt. of CP *CP = Crude powder; CAME = Crude aqueous methanol extract

25 Table 9: Layout plan for crude powder and crude aqueous methanolic extract of Areca catechu (seeds) and Ferula assa-foetida (latex) given to different groups of sheep naturally infected with mixed species of gastrointestinal nematodes Groups Treatment 1 Untreated control 2 Levamisole HCl (Nilverm® 1.5%, w/v; ICI Pakistan Limited, Animal Health Division) at 7.5 mg kg−1 body weight (b.wt.) 3 Crude powder (CP) at 0.33 g kg−1 b.wt. 4 CP at 0.66 g kg−1 b.wt. 5 CP at 1 g kg−1 b.wt. 6 Crude aqueous methanolic extract (CAME) at equivalent dose rate 0.33 g kg−1 b.wt. of CP 7 CAME at the equivalent dose rate 0.66 g kg−1 b.wt. of CP 8 CAME at the equivalent dose rate 1 g kg−1 b.wt. of CP *CP = Crude powder; CAME = Crude aqueous methanol extract (iii) Measurements Fecal egg counts were performed on each animal on days 0, 4, 8 and 12 post-treatment. 3.3. Statistical Analyses Data from egg hatch test were transformed by probit transformation against the logarithm of plant extract (Hubert and Kerboeuf, 1992). Probit transformation was performed to transform a typical sigmoid curve dose response to a linear function. The lethal concentration 50

(LC50) of extract concentration, required to prevent 50% hatching of eggs (in case of egg hatch test) was calculated from the linear regression (for y = 0 on the probit scale). In adult motility assay, comparison between means of dead worms was made using DMR Test. For in vivo studies, egg count percent reduction (ECR) was calculated using the following formula: ECR (%) = (Pre treatment egg count per gram ˗ Post treatment egg count per gram/Pre treatment egg count per gram) x 100 The results were expressed as eggs per gram (Mean+SEM) of feces and means were compared by using DMR Test (SAS, 1998).

26 Chapter # 4 Results The present study was conducted: (i) To document the Ethno-veterinary Practices (EVM) for the treatment of different ailments of animals in some parts of District Jhang (Punjab), Pakistan (ii) To validate the use of some plants as anthelmintics employing standard parasitological procedures. 4.1. Documentation of EVM practices EVM practices used for the treatment and/or control of different ailments/conditions/disorders documented in this study were based on plants, materials of animal origin and others including salts, elements, etc. and are listed in the following tables. A total of 46 plants were documented (Table 10) for their use in different ailments/conditions of animals. Of the total documented, 33 plants were indigenous to the area. However, some of these plants (e.g., Ferula assa-foetida Linn., Amomum subulatum Roxb., Areca catechu Linn., and Piper nigrum Linn.) were purchased from herbal shops in the nearby towns. Table 10: Botanical, local and English names of the plants documented from district Jhang (Punjab, Pakistan) for their use in ethno veterinary medicine

S. Botanical Name of Plant* Local Name English Voucher Indigenous/Non- No. Name No. indigenous 1. Acacia nilotica (Linn.) Delile, Fl.Aegypt. Desi Kikar Babul tree Jg1/2006 Indigenous 2. Allium cepa Linn. Pyaaz Onion Jg2/2006 Indigenous 3. Allium sativum Linn. Thoam Garlic Jg3/2006 Indigenous 4. Aloe vera (L.) Burm.f. Kanwar gandal Indian aloes Jg4/2006 Indigenous 5. Chenopodium album (L.) Bathu Common Jg5/2006 Indigenous lamb's quarter 6. Amomum subulatum Roxb Bari Ilaichi Greater Jg6/2006 Non-indigenous cardamom 7. Areca catechu Linn. Supari Betel nut Jg7/2006 Non-indigenous palm 8. Arundo donax Linn. Nara Giant reed Jg8/2006 Indigenous 9. Azadirachta indica A. Juss. Neem Persian lilac Jg9/2006 Indigenous 10. Brassica campestris L. Sersoon Mustard Jg10/2006 Indigenous 11. Butea frondosa Roxb. Kamar kas Flame of the Jg11/2006 Non-indigenous Forest 12. Calotropis procera (Ait.) R.Br. Aak Mudar Jg12/2006 Indigenous 13. Cannabis sativa Linn. Bhang Wild hemp Jg13/2006 Non-indigenous 14. Capsicum annum Linn. Lal mirch Spanish Jg14/2006 Indigenous pepper 15. Citrullus colocynthis (Linn.) Schrad. Kor tunbah Bitter apple Jg15/2006 Indigenous 16. Citrus limon (L.) Burm.f. Nimbo Lemon Jg16/2006 Indigenous

17. Commiphora wightii (Arn.) Bhandari Gugal Indian Jg17/2006 Indigenous Bdellium Tree 18. Cucurbita maxima Duch. ex Lam. Kadu Red Gourd Jg18/2006 Indigenous 19. Cuminum cyminum Linn. Safed zera Cumin seed Jg19/2006 Non-indigenous 20. Curcuma longa Linn. Haldi Turmeric Jg20/2006 Non-indigenous 21. Dalbergia sissoo Roxb. Ex DC. Tali Indian Jg21/2006 Indigenous Rosewood.

27 Table 10 Contd.. S. Botanical Name of Plant* Local Name English Voucher Indigenous/Non- No. Name No. indigenous 22. Embelia ribes Burm. F. Babrung Embelia Jg22/2006 Non-indigenous http://www.eco- planet.com/Herbsandplants/EmbeliaribesBurm.f..htm 23. Eugenia jambolana Lam. Jamoon Black berry Jg23/2006 Indigenous 24. Ferula assa-foetida Linn. Heang Asafoetida Jg24/2006 Non-indigenous 25. Ficus religiosa Linn. Pepal Sacred fig Jg25/2006 Indigenous 26. Foeniculum vulgare Mill. Sounf Fennel Jg26/2006 Indigenous 27. Fumaria indica (Hausskn.) Papara Fumitory Jg27/2006 Indigenous 28. Linum usitatissimum Linn. Alsi Linseed Jg28/2006 Indigenous 29. Mallotus philippensis (Lam.) Muell. Arg. Kamala Rottlera Jg29/2006 Indigenous 30. Mentha arvensis Linn. Poodina Corn mint Jg30/2006 Indigenous 31. Morus alba L. Toot White Jg31/2006 Indigenous Mulberry 32. Nicotiana tabacum L Tambaco Flowering Jg32/2006 Indigenous Tobacco 33. Papaver somniferum L. Post Opium poppy Jg33/2006 Non-indigenous 34. Piper nigrum Linn. Kali Mirch Black pepper Jg34/2006 Non-indigenous 35. Psidium guajava L. Amrood Guava Jg35/2006 Indigenous 36. Punica granatum L. Anar Pomegranate Jg36/2006 Indigenous 37. Raphanus sativus Linn. Mooli Radish Jg37/2006 Indigenous 38. Lepidium sativum Linn Halu Garden cress Jg38/2006 Indigenous 39. Solanum melongena L. Bengan Brinjal Jg39/2006 Indigenous 40. Tamarindus indica L. Imli Tamarind Jg40/2006 Indigenous 41. Trachyspermum ammi L. Sprague Ajwain porji Omum Jg41/2006 Indigenous 42. Vernonia anthelmintica L. Kali Zeeri Purple Jg42/2006 Non-indigenous fleebane 43. Vitex negundo L Smalu Cut-Leaf Jg43/2006 Indigenous Chastetree 44. Withania coagulans Dunal. Paneer Vegetable Jg44/2006 Indigenous rennet 45. Zingiber officinale Roscoe Adrak Ginger Jg45/2006 Indigenous 46. Ziziphus jujuba Mill. Bair Jujube Jg46/2006 Indigenous *Botanical names as given by Kapoor (2001), Williamson (2002) and on www.eFloras.org The documented plants represented 31 families (Table 11). Solanaceae and Umbelliferae was the most represented family by four plants followed by Brassicaceae and Zingiberaceae (each by three plants), Cucurbitaceae, Liliaceae, Moraceae and Myrtaceae (each by two plants) and Asclepiadaceae, Asteraceae, Burseraceae, Cannabaceae, Chenopodiaceae, Euphorbiaceae,

Fabaceae (Papilionoideae), Fumariaceae, Labiatae, Linaceae, Meliaceae, Myrsinaceae, Palmae Papaveraceae, Papilionaceae, Piperaceae, Poaceae, Punicaceae, Rhamnaceae, Rutaceae, and Verbenaceae (each by one plant). Table 11: Name of plants, representing families and frequency of their usage in ethno veterinary medicine in district Jhang (Punjab, Pakistan)

Representing Plant Families Name of Plants Respondents Frequency (Out of 253) (%) Families representing four plants Solanaceae Capsicum annum Linn. 96 37.94 Solanaceae Nicotiana tabacum L. 13 5.13 Solanaceae Solanum melongena L. 30 11.85 Solanaceae Withania coagulans Dunal. 33 13.04 Umbelliferae Cuminum cyminum Linn. 52 20.55 Umbelliferae Ferula assa-foetida Linn. 78 30.83 Umbelliferae Foeniculum vulgare Mill. 84 33.20 Umbelliferae Trachyspermum ammi (L.) Sprague. 175 69.16

28 Table 11 Contd. Representing Plant Families Name of Plants Respondents Frequency (Out of 253) (%) Families representing three plants Brassicaceae Brassica campestris L. 13 51.38 Brassicaceae Raphanus sativus Linn. 21 8.3 Brassicaceae Lepidium sativum Linn. 22 8.69 Zingiberaceae Amomum subulatum Roxb 58 22.92 Zingiberaceae Curcuma longa Linn. 4 1.58 Zingiberaceae Zingiber officinale Roscoe 13 5.13 Families representing two plants Cucurbitaceae Cucurbita maxima Duch. ex Lam. 53 20.94 Cucurbitaceae Citrullus colocynthis (Linn.) Schrad. 24 9.48 Liliaceae Allium cepa Linn. 83 32.80 Liliaceae Allium sativum Linn. 33 13.04 Moraceae Ficus religiosa Linn. 14 5.53 Moraceae Morus alba L. 12 4.74 Myrtaceae Eugenia jambolana Lam. 13 5.13 Myrtaceae Psidium guajava L. 4 1.58 Families representing one plant Asclepiadaceae Calotropis procera (Ait.) R.Br.* 32 12.64 Asphodelaceae Aloe vera (L.) Burm. f. 25 9.88 Asteraceae Vernonia anthelmintica L. 95 35.54 Burseraceae Commiphora wightii (Arn.) Bhandari 17 6.71 Cannabaceae Cannabis sativa Linn.. 37 14.62 Chenopodiaceae Chenopodium album (L.) 8 3.16 Euphorbiaceae Mallotus philippensis (Lam.) Muell. Arg. 24 9.48 Fabaceae (Papilionoideae) Dalbergia sissoo Roxb. Ex DC. . 20 7.90 Fumariaceae Fumaria indica (Hausskn.) 8 3.16 Labiatae Mentha arvensis Linn. 46 18.18 Leguminosae Tamarindus indica L. 59 23.32 Linaceae Linum usitatissimum Linn. 47 18.57 Meliaceae Azadirachta indica A. Juss. 36 14.22 Mimosaceae Acacia nilotica (Linn.) Delile, Fl.Aegypt. 35 13.83 Myrsinaceae Embelia ribes Burm. F. 12 4.74 Palmae Areca catechu Linn. 24 9.48 Papaveraceae Papaver somniferum L. 12 4.74 Papilionaceae Butea frondosa Roxb. 8 3.16 Piperaceae Piper nigrum Linn. 80 31.62 Poaceae Arundo donax Linn. 8 3.16 Punicaceae Punica granatum L. 33 13.04 Rhamnaceae Ziziphus jujuba Mill. 24 9.48 Rutaceae Citrus limon (L.) Burm.f. 55 21.73 Verbenaceae Vitex negundo L. 8 3.16

The top ten most frequently reported (≥ 22% respondents) plants for their usage in EVM included Trachyspermum ammi (L.) Sprague., Capsicum annum Linn., Vernonia anthelmintica L., Foeniculum vulgare Mill, Allium cepa Linn, Piper nigrum Linn, Ferula assa-foetida Linn., Tamarindus indica L., Amomum subulatum Roxb and Citrus limon (L.) Burm.f. (Table 12). Table 12: Top ten most frequently reported plants for their usage in ethno veterinary medicine in Jhang (Punjab, Pakistan)

Representing Plant Families Name of Plants Respondents Frequency (Out of 253) (%) Apiaceae Trachyspermum ammi (L.) Sprague. 175 69.16 Solanaceae Capsicum annum Linn. 96 37.94 Asteraceae Vernonia anthelmintica L. 95 35.54 Apiaceae Foeniculum vulgare Mill. 84 33.20 Liliaceae Allium cepa Linn. 83 32.80 Piperaceae Piper nigrum Linn. 80 31.62 Umbelliferae Ferula assa-foetida Linn. 78 30.83 Leguminosae Tamarindus indica L. 59 23.32 Zingiberaceae Amomum subulatum Roxb. 58 22.92 Rutaceae Citrus limon (L.) Burm.f. 55 21.73

29 Materials other than plants used as vehicle or as a remedy included tartaric acid, molasses, ammonium chloride, jaggery, sodium chloride, sodium bicarbonate, water, sugar, butter and butter fat. EVM practices for different diseases/conditions of livestock reported by the respondents in Jhang are given in Table 13. Table 13: Inventory of plants used for the treatment of different diseases/conditions of livestock reported by the local respondents in Jhang (Punjab, Pakistan) No. Indication/Plants/ Mode of use Respondents Practices used (out of 250) Aglactia 1 Linum usitatissimum Linn. 60-100 g seeds mixed with 20 g tartaric acid and 250 9 g molasses; Given PO for three days 2 Amomum subulatum Roxb. 50 g powdered seeds mixed with 50 g of ammonium 9 chloride and 250 g molasses; Given PO as physic drench ball 3 Citrus limon (L.) Burm.f. 200-300 g fruit mixed with 500 g molasses; Given 9 PO for three days 4 Cuminum cyminum Linn. Give 60 g seed powder PO 5 5 Lepidium sativum Linn. + Brassica 250 g seeds of Lepidium sativum mixed with 100 mL 9 campestris L. Brassica oil and 250 g of molasses; Given PO 6 Brassica campestris L. 250-500 mL seed oil PO for three alternate days 5 7 Tamarindus indica L. 200-300 g fruit with 500 g jaggery; Given PO 5 Total entries 51 Allergy 1 Vernonia anthelmintica L. 60-100 g seeds with 250 g molasses; Given PO 13 2 Capsicum annum Linn. 250 g fruit; Given PO 13 3 Piper nigrum Linn. 50-100 g pepper corns with 250 g molasses; Given 13 PO as physic drench ball Total entries 39 Anestrous 1 Capsicum annum Linn. 250 g fruit; Given PO 21 2 Trachyspermum ammi (L.) 50-100 g powdered Trachyspermum ammi seeds 21 Sprague. + Allium cepa Linn. mixed with 250 g Allium cepa bulbs and added 50 g each sodium chloride and molasses; Given PO 3 Solanum melongena L. 500 g half cooked fruit; Given PO 17 Total entries 59 Anorexia 1 Punica granatum L. + Mentha Mix 250 g Punica granatum fruit, 200 g Allium cepa 29 arvensis Linn. + Allium cepa bulbs, and 100 g Mentha arvensis seeds with 50 g Linn. each sodium chloride and sodium bicarbonate; Give PO as physic drench ball 2 Amomum subulatum Roxb. 50 g powdered seeds with 30 g ammonium chloride 16 and 250 g molasses; Given PO 3 Zingiber officinale Roscoe 50-150 g root powder with 250 g molasses; Given 13 PO Total entries 58 Chronic Diarrhea 1 Nicotiana tabacum L. Two liters of tobacco smoked water PO 13 2 Eugenia jambolana Lam. Give 5kg leaves as feed stuff; Given PO 5 Total entries 18

30 Table 13 Contd. No. Indication/Plants/ Mode of use Respondents Practices used (out of 250) Colic 1 Ferula assa-foetida Linn. 25-50 g resin with 250 g jaggery; Given PO 17 2 Trachyspermum ammi (L.) 50-100 g powdered Trachyspermum ammi seeds and 17 Sprague. + Mentha arvensis Linn. 200 g Mentha arvensis leaves with 250 g molasses; Given PO Total entries 34 Constipation 1 Fumaria indica (Hausskn.) Give 5 kg fresh leaves as feed stuff PO 8 2 Solanum melongena L. 500 g half cooked fruit; Given PO 13 3 Trachyspermum ammi (L.) 50 g sodium chloride and 100 g powdered 13 Sprague. Trachyspermum ammi seeds mixed with 500 g jaggery; Given PO Total entries 34 Fever 1 Trachyspermum ammi (L.) 50-100 g powdered seeds with 250 g molasses; 13 Sprague. Given PO Total entries 13 Foot and Mouth Disease 1 Camel wool Make a locket and hang in neck 4 2 Acacia nilotica (Linn.) Delile, 500 g bark and 50 g sodium chloride boiled in water; 13 Fl.Aegypt. Used topically for hoof wound dressing 3 Aloe vera (L.) Burm. f. 100-150 g powdered leaves mixed with 250 g 13 jaggery; Given PO Total entries 30 Foot rot 1 Acacia nilotica (Linn.) Delile, 500 g bark and 50 g sodium chloride boiled in water; 13 Fl.Aegypt. Used topically for wound dressing Total entries 13 Galactagogue 1 Citrus limon (L.) Burm.f. 200-300 g fruit with 250g molasses; Given PO 13 2 Linum usitatissimum Linn. + 60-100 g Linum usitatissimum seeds mixed with 500 13 Foeniculum vulgare Mill. + g molasses, 100 g Foeniculum vulgare seeds and 250 Lepidium sativum Linn. g Lepidium sativum seeds; Given PO 3 Tamarindus indica L. 200-300 g fruit mixed with 250 g jaggery; Given PO 9 4 Cuminum cyminum Linn. 60 g seeds powder; Given PO 9 5 Cannabis sativa Linn. 100-250 g powdered leaves mixed with 250 g 9 jaggery; Given PO Total entries 53 Hemorrhagic septicemia 1 Morus alba L. + Curcuma longa 500 g Morus alba leaves mixed with 250 g Curcuma 4 Linn. + Brassica campestris L. longa rhizomes and 250 mL Brassica oil; Make paste and apply tropically on throat 2 Ficus religiosa Linn. + Brassica 2-3 kg Ficus religiosa leaves and 250 mL Brassica 4 campestris L. oil boiled in 1 L water; Make a paste and apply topically Total entries 8 Heat stroke 1 Morus alba L. 500 g fruit mixed with 250 g sugar and 1 L water; 4 Given PO 2 Tamarindus indica L. 200-300 g fruit mixed with 250 g jaggery; Given PO 4 3 Cuminum cyminum Linn. 60 g seeds powder; Given PO 4 Total entries 12

31 Table 13 Contd.. No. Indication/Plants/ Mode of use Respondents Practices used (out of 250) Indigestion 1 Trachyspermum ammi (L.) 50 g each sodium chloride and ammonium chloride 54 Sprague. + Allium cepa Linn. + mixed with 250 g Allium cepa bulb, 60 g Foeniculum vulgare Mill. Foeniculum vulgare seeds and 100 g powdered Trachyspermum ammi seeds; Given PO Total entries 54 Traumatic inflammation 1 Vernonia anthelmintica L.. 60-100 g seeds mixed with 250 g molasses; Given 21 PO 2 Capsicum annum Linn. 250 g fruit; Given PO 17 3 Piper nigrum Linn. 50-100 g pepper corns; Given PO 21 4 Commiphora wightii (Arn.) 100 g leaves; Given PO 17 Bhandari. Total entries 55 Non Specific Jaundice 1 Tamarindus indica L. 200-300 g fruit mixed with jaggery; Given PO 8 2 Citrus limon (L.) Burm.f. 200-300 g fruit mixed with molasses; Given PO 8 3 Cuminum cyminum Linn. 60 g seeds powder; Given PO 8 Total entries 24 Lochia remover 1 Trachyspermum ammi (L.) 50-100 g powdered seeds mixed with molasses; 17 Sprague. Given PO 2 Foeniculum vulgare Mill. 100 g powdered seeds mixed with 250 g molasses; 17 Given PO 3 Linum usitatissimum Linn. 60-100 g seeds mixed with 250 g molasses; Given 17 PO 4 Amomum subulatum Roxb. 50 g powdered seeds mixed with 50 g each sodium 17 chloride, and ammonium chloride and 250 g molasses; Given PO Total entries 68 Mastitis 1 Vernonia anthelmintica L. 60-100 g seeds mixed with 250 g molasses; Given 37 PO 2 Tamarindus indica L. 200-300 g fruit mixed with 250 g jaggery; Given PO 21 3 Citrus limon (L.) Burm.f. 200-300 g fruit mixed with 250 g molasses; Given 25 PO 4 Allium sativum L. 250-500 g half cooked bulbs mixed with 500 g 33 sugar; Given PO 5 Ferula assa-foetida Linn. 20 g half cooked latex mixed with 500 g sugar; 13 Given PO 6 Capsicum annum Linn. 250 g fruit; Given PO 29 7 Piper nigrum Linn. 50-100 g pepper corns fried in butter; Given PO 46 8 Withania coagulans Dunal. Give 100-150 g aerial parts in jaggery and give PO 21 Total entries 225 Panting 1 Tamarindus indica L. 200-300 g fruit mixed with jaggery; Given PO 4 2 Cuminum cyminum Linn. 60 g seeds powder; Given PO 4 3 Cannabis sativa Linn.. 100-250 g powdered leaves mixed with 250 g 4 jaggery; Given PO Total entries 12 Pneumonia 1 Morus alba L 1 kg fruit mixed with 1 kg sugar and boiled in 2 L 4 water; Given 500 mL daily PO Total entries 4

32 Table 13 Contd.. No. Indication/Plants/ Mode of use Respondents Practices used (out of 250) Prolapse 1 Cannabis sativa Linn.. 100-250 g powdered leaves mixed with jaggery; 12 Given PO 2 Papaver somniferum L. 20 g latex mixed with 250 g molasses; Given PO 12 3 Dalbergia sissoo Roxb. Ex DC. 2 kg leaves boiled in 2 L water; Given 500 mL daily 20 PO for three days 4 Cucurbita maxima Duch. ex Lam. 50-150 g seed powder mixed with 250 g jaggery; 16 Given PO 5 Ziziphus jujuba Mill. l kg leaves bolied in 1 L water; Given PO 12 Total entries 72 Red water 1 Cuminum cyminum Linn. 60 g seed powder; Given PO 21 2 Cannabis sativa Linn. 100-250 g powdered leaves mixed with jaggery; 12 Given PO 3 Cucurbita maxima Duch. ex Lam. 50-150 g seed powder mixed with 250 g jaggery; 21 Given PO 4 Tamarindus indica L. 200-300 g fruit mixed with 250g jaggery; Given PO 8 5 Raphanus sativus Linn. Fresh 5-10 kg roots; Given PO 21 Total entries 83 Rheumatism 1 Linum usitatissimum Linn. 60-100 g seeds mixed with 250 g molasses; Given 8 PO 2 Vernonia anthelmintica L. 60-100 g seeds mixed with 250 g molasses; Given 8 PO Total entries 16 Toxemia 1 Calotropis procera (Ait.) R.Br.* 50-200 g flowers mixed with 250 g jaggery; Given 24 PO 2 Vernonia anthelmintica L. 60-100 g seeds mixed with 250 g molasses; Given 16 PO 3 Capsicum annum Linn. 250 g fruit; Given PO 16 4 Azadirachta indica A. Juss. 500 g leaves mixed with 250 g molasses; Given PO 8 5 Aloe vera (L.) Burm. f. 100-150 g leaves mixed with 250 g jaggery; Given 12 PO Total entries 76 Tympany 1 Ferula assa-foetida Linn. + 100 g Trachyspermum ammi seeds mixed with 60 g 28 Trachyspermum ammi (L.) sodium bicarbonate, and 125 g molasses; Given PO Sprague. 2 Citrullus colocynthis (Linn.) 100-250 g dried fruit powder mixed with 250 g 24 Schrad. jaggery; Given PO Total entries 52 External wounds 1 Azadirachta indica A. Juss. 500 g leaves and 50 g sodium chloride boiled in 2 L 20 water; Use topically for wound dressing 2 Ziziphus jujuba Mill. l kg leaves bolied in 2 L water; Used topically for 12 wound dressing Total entries 32 Worm infestation 1 Mallotus philippensis (Lam.) 50-100 g fruit mixed with 250 g jaggery; Given PO 24 Muell. Arg. 2 Areca catechu Linn. 50-100 g seeds mixed with jaggery; Given PO 24 3 Ferula assa-foetida Linn. 25-50 g latex mixed with 250 g jaggery; Given PO 20

33 Table 13 Contd. No. Indication/Plants/ Mode of use Respondents Practices used (out of 250) 4 Amomum subulatum Roxb. 25-50 g powdered seeds mixed with 250 g jaggery; 16 Given PO 5 Cucurbita maxima Duch. ex Lam. 50-150 g seed powder; Given PO 16 6 Trachyspermum ammi (L.) 50-100 g powdered seeds mixed with 250 g jaggery; 12 Sprague. Given PO 7 Embelia ribes Burm. F. 100-150 g seed powder mixed with 250 g jaggery; 12 Given PO 8 Withania coagulans Dunal. 50-200 g aerial parts mixed with 250 g jaggery; 12 Given PO 9 Calotropis procera (Ait.) R.Br.* 50-200 g flowers mixed with 250 g jaggery; Given 8 PO 10 Arundo donax Linn. 100-250 g fresh leaves; Given PO 8 11 Azadirachta indica A. Juss. 500 g leaves mixed with 250 g molasses; Given PO 8 12 Butea frondosa Roxb. 100-250 g powdered leaves mixed with 250 g 8 jaggery; Given PO 13 Chenopodium album (L.) 5 kg green leaves; Given PO 8 14 Vitex negundo L. 100-250 g powdered seeds mixed with 250 g 8 jaggery; Given PO 15 Psidium guajava L. 50-150 g seed powder; Given PO 4 16 Punica granatum L. 250-300 g fruit peel boiled in 1 L water; Given PO 4

17 Acacia nilotica (Linn.) Delile, 100 g fresh leaves mixed with butter fat; Given PO 4 Fl.Aegypt. 18 Nicotiana tabacum L Two liters of tobacco smoked water PO 2 19 Papaver somniferum L. Give 200 g as decoction 1 Total entries 199 PO= Per os There was maximum usage of seeds (n=37) followed by fruit (n=23), leaves (n=22), bulb (n=4), latex and pepper corns (n=3 each), aerial parts, bark, flowers and roots (n=2 each), fruit peel, resin and rhizomes (n=1 each). The vehicles/adjuncts used were jaggery (n=30), molasses (n=28), sodium chloride (n=8), sugar and ammonium chloride (n=4), sodium bicarbonate (n=2), and butter, butter fat and tartaric acid (n=1 each). The recipes were used as powder, decoction, oil and paste. The major mode of administration/application was per os (n=93) followed by topical application (n=7), tobacco smoked water (n=2) and hanging in the neck as a locket (n=1). Number and nature of EVM practices documented for the treatment of different diseases/conditions of livestock in Jhang have been presented in Table 14. The maximum number (n=225) of entries as far as usage of traditional remedies is concerned was for Mastitis. This was followed in decreasing order by worm infestation (n=199), Red water (n=83), Toxemia (n=76), Prolapse (n=72), Lochia remover (n=68), Anestrous (n=59), Anorexia (n=58), Traumatic inflammation (n=55), Indigestion (n=54), Galactagogue (n=53), Tympany (n=52), Aglactia (n=51), Allergy (n=39), Chronic Diarrhea (n=18) Constipation (n=34), Colic (n=34), External wounds (n=32), Foot and Mouth Disease (n=30),

34 Non-specific jaundice (n=24), Rheumatism (n=13), Fever and Foot rot (n=13), Heat stroke and Panting (n=12), Hemorrhagic septicemia (n=8) and Pneumonia (n=4). EVM practices varied in source and/or form of the plant/material used, combination with other plants/materials, vehicles and mode of preparation and administration/application. On an overall, 102 EVM practices were recorded. Table 14: Number and nature of EVM practices for different diseases/conditions documented from Jhang (Punjab, Pakistan)

Disease/condition No. of No. of No. of other No. of Use of plants/materials Variation in case of use remedies plants Materials entries more than one time more than one time Worm infestation 19 19 - 199 - - Mastitis 8 8 - 225 - - Aglactia 7 7 - 51 Brassica campestris (02) Alone and in combination with other plants Galactagogue 5 7 - 53 - - Prolapse 5 5 - 72 - - Red water 5 5 - 83 - - Toxemia 5 5 - 76 - - Traumatic inflammation 4 4 - 55 - - Lochia remover 4 4 - 68 - - Allergy 3 3 - 39 - - Anestrous 3 4 - 59 - - Anorexia 3 5 - 58 - - Constipation 3 3 - 34 - - Foot and Mouth Disease 3 2 1 30 - - Heat stroke 3 3 - 12 - - Non-specific jaundice 3 3 - 24 - - Panting 3 3 - 12 - - Chronic Diarrhea 2 2 - 18 - - Colic 2 3 - 34 - - Hemorrhagic septicaemia 2 4 - 8 Brassica campestris (02) In different combinations Rheumatism 2 2 - 13 - - Tympany 2 3 - 52 - - External wounds 2 2 - 32 - - Fever 1 1 - 13 - - Foot rot 1 1 - 13 - - Indigestion 1 3 - 54 - - Pneumonia 1 1 - 4 - - Total 102 112 1 1373 - -

Plants, diversity of their usage and contribution in total number of EVM practices for diseases/conditions have been presented in Table 15. A total 46 plant species were documented for their use in EVM practices. Twenty six plant species were used for the treatment of more than one disease/condition. Trachyspermum ammi L. Sprague. was the most diversely used plant (in eight diseases/conditions), followed by Tamarindus indica L. (in seven diseases/conditions), Cuminum cyminum Linn. (in six diseases/conditions), Capsicum annum Linn. and Vernonia anthelmintica L. (each in five diseases/conditions), Amomum subulatum Roxb., Cannabis sativa Linn., Citrus limon (L.) Burm. f., Ferula assa-foetida Linn. and Linum usitatissimum Linn. (each in four diseases/conditions), Acacia nilotica (Linn.) Delile, Fl.Aegypt., Allium cepa Linn., Azadirachta indica A. Juss., Brassica campestris L., Cucurbita maxima Duch. ex Lam., Foeniculum vulgare Mill., Morus alba L., and Piper nigrum Linn. (each in three

35 diseases/conditions), and Aloe vera (L.) Burm. f., Calotropis procera (Ait.) R.Br., Mentha arvensis Linn., Nicotiana tabacum L., Punica granatum L., Lepidium sativum Linn., Solanum melongena L., Withania coagulans Dunal. and Ziziphus jujuba Mill. (each in two diseases/conditions). Table 15: Plants, diversity of their usage and contribution in total number of EVM practices for different diseases/conditions of livestock in Jhang (Punjab, Pakistan) Plants Usage diversity Part of EVM Respondents practices (out of 250) (No.) 1. Trachyspermum ammi (L.) CP+FR+LR+WI+ANE+IDG+CO+TY 8 158 Sprague. 2. Tamarindus indica L. AG+GG+HST+NSJ+MST+PG+RW 7 59 3. Cuminum cyminum Linn. AG+GG+HST+NSJ+PG+RW 6 51 4. Capsicum annum Linn. AL+ANE+TI+MST+TM 5 96 5. Vernonia anthelmintica L. AL+TI+MST+RT+TM 5 95 6. Amomum subulatum Roxb. AG+AN+LR+WI 4 58 7. Cannabis sativa Linn.. GG+PG+PL+RW 4 37 8. Citrus limon (L.) Burm.f. AG+GG+NSJ+MST 4 55 9. Ferula assa-foetida Linn. CO+MST+WI+TY 4 78 10. Linum usitatissimum Linn. AG+LR+RT+GG 4 47 11. Acacia nilotica (Linn.) FMD+FR+WI 3 30 Delile, Fl.Aegypt. 12. Allium cepa Linn. AN+ IDG+ANE 3 104 13. Azadirachta indica A. Juss. TM+EW+WI 3 36 14. Brassica campestris L. AG+HS+AG 3 22 15. Cucurbita maxima Duch. ex PL+RW+WI 3 53 Lam. 16. Foeniculum vulgare Mill. LR+IDG+GG 3 74 17. Morus alba L PN+HST+HS 3 12 18. Piper nigrum Linn. AL+TI+MST 3 80 19. Aloe vera (L.) Burm. f. FMD+TM 2 25 20. Calotropis procera (Ait.) TM+WI 2 32 R.Br.* 21. Mentha arvensis Linn. AN+CO 2 46 22. Nicotiana tabacum L CD+WI 2 13 23. Punica granatum L. WI+AN 2 33 24. Lepidium sativum Linn. AG+GG 2 22 25. Solanum melongena L. ANE+CP 2 30 26. Withania coagulans Dunal. MST+WI 2 33 27. Ziziphus jujuba Mill. PL+EW 2 24 28. Allium sativum L. MST 1 33 29. Chenopodium album (L.) WI 1 8 30. Areca catechu Linn. WI 1 24 31. Arundo donax Linn. WI 1 8 32. Butea frondosa Roxb. WI 1 8 33. Citrullus colocynthis (Linn.) TY 1 24 Schrad. 34. Commiphora wightii (Arn.) TI 1 17 Bhandari . 35. Curcuma longa Linn. HS 1 4

36 Table 15 Contd. Plants Usage diversity Part of EVM Respondents practices (out of 250) (No.) 36. Dalbergia sissoo Roxb. Ex PL 1 20 DC. 37. Embelia ribes Burm. F. WI 1 12 38. Ficus religiosa Linn. HS 1 4 39. Fumaria indica (Hausskn.) CP 1 8 40. Mallotus philippensis (Lam.) WI 1 24 Muell. Arg. 41. Eugenia jambolana Lam. CD 1 13 42. Papaver somniferum L. PL 1 12 43. Psidium guajava L. WI 1 4 44. Raphanus sativus Linn. RW 1 21 45. Vitex negundo L. WI 1 8 46. Zingiber officinale Roscoe AN 1 13 AG= Aglactia; AL= Allergy; ANE= Anestrous; AN= Anorexia; CD=Chronic Diarrhea; CO= Colic; CP= Constipation; EW= External wounds; FR= Fever; FMD= Foot and Mouth Disease; FR= Foot rot; GG= Galactagogue; HS= Hemorrhagic septicaemia; HST= Heat stroke; IDG= Indigestion; LR= Lochia remover; MST= Mastitis; NSJ= Non Specific Jaundice; PG= Panting; PN= Pneumonia; PL= Prolapse; RW= Red water; RT= Rheumatism; TM= Toxemia; TI= Traumatic inflammation; TY= Tympany; WI= Worm infestation

Salient findings of the study on EVM documentation 1. Of the total number of EVM practices reported by the respondents, remedies for worm infestation had the maximum contribution (≈17%). 2. Forty six plants were documented for their use in EVM practices in Jhang (Punjab, Pakistan) indicating an important role of plants in the treatment of different diseases of livestock. 3. There was wide variation in the dose, vehicle, form of plant used, mode of preparation and administration/application for the use of plants even among the EVM practices for the same disease/condition 4. There was wide diversity in usage and in combination of different plants for the treatment of different diseases.

37 4.2. In vitro and in vivo anthelmintic activity The yield of extraction with different solvents is given as under: Plant Weight before Weight before dipping Percent yield dipping (kg) (g) Acacia nilotica leaves 05 1297.6 26.0 Acacia nilotica bark 05 439.2 17.6 Amomum subulatum 05 481.2 9.6 Ferula assafoetida 01 407.1 40.7 Vitex negundo 05 235.7 4.7 Arundo donax 05 350.3 7.0 Arecha catechue 05 999.2 20.0 Yield (g) of fractions *CAME of plant Aqueous (g) Chloroform (g) Ethyle acetate Petroleum spirit Acacia nilotica leaves 7.96 5.85 0.57 0.42 Acacia nilotica bark 8.0 0.5 0.52 0.45 Amomum subulatum 3.98 0.72 0.42 1 Ferula assafoetida 10.38 4.68 6.27 0.62 Vitex negundo 10.5 0.5 0.78 0.57 Arundo donax 11.5 0.44 0.41 5.0 Arecha catechue 17.02 0.92 0.8 1.2 *CAME= Crude aqueous methanol extract (20 g of each plant)

In vitro (adult motility assay and egg hatch assay) and in vivo (fecal egg count reduction test) tests were carried out to evaluate the anthelmintic activity of selected plants. The results are given in the following sections. 4.2.1. Adult Motility Assay The criteria for interpretation of results of adult mortality assay in this study were (i) hours taken for mortality of 100% worms (H. contortus), and (ii) dose dependent response of worms to crude extracts of different plants. All the plants included in this study exhibited anthelmintic activity against H. contortus. A wide variation, however, was recorded in the anthelmintic effects among different plants and fractions of plant extracts (Table 16-23). In the order of decreasing efficacy, crude aqueous methanol extracts (CAME) of Ferula assa-foetida, Acacia nilotica bark, Areca catechu, Amomum subulatum, Vitex negundo, Acacia nilotica leaves and Arundo donax exerted anthelmintic effects (P ≥ 0.05) @ 25-50 mg mL-1. There was 100% mortality of worms in Levamisole (used as a reference drug) within 2 hours post-exposure. Dose and time dependent anthelmintic effects were recorded for all the plants; however, by 10 hours post-exposure, 100% mortality of worms was recorded only for Ferula assa-foetida @ 12.5-50 mg mL-1 and Acacia nilotica bark @ 50 mg mL-1 (Table 16). Results of different fractions of plants are given in Table 24. Ethyle acetate was found to be the most effective fraction of all the plants included in the study followed in general by chloroform (except Vitex negundo). The petroleum spirit and aqueous fractions of all the plants were least effective.

38 Table 16: In vitro effect of crude aqueous methanol extracts of different plants on survival of H. contortus of sheep in comparison with Levamisole

Treatments Number of dead worms (Mean±SE) at different hours Hours post-exposure 0 hr 2 hr 6 hr 10 hr Levamisole 0.5 mg mL-1 0.00 ±0.00n 10.00±0.00a 10.00±0.00a 10.00±0.00a PBS 0.00 ±0.00n 0.00 ±0.00n 0.00 ±0.00n 0.00 ±0.00n Ferula assa-foetida 1.56 mg mL-1 0.00±0.00n 0.00 ±0.00n 1.00±0.000mn 7.33±0.333de 3.12 mg mL-1 0.00±0.00n 0.00±0.00n 1.67±0.333klm 7.00±0.577de 6.25 mg mL-1 0.00±0.00n 0.00±0.00n 2.33±0.333jkl 8.67±0.333abc 12.5 mg mL-1 0.00±0.00n 0.67±0.333mn 4.33±0.882gh 10.00±0.000a 25 mg mL-1 0.00±0.00n 3.33±0.333hij 6.33±0.882ef 10.00±0.000a 50 mg mL-1 0.00±0.00n 5.67±0.333f 8.00±0.577bcd 10.00±0.000a Acacia nilotica bark 1.56 mg mL-1 0.00±0.00o 0.00±0.00o 0.33±0.333no 1.33±0.333kn 3.12 mg mL-1 0.00±0.00o 0.00±0.00o 0.67±0.333mno 1.67±0.333jm 6.25 mg mL-1 0.00±0.00o 0.33±0.333no 1.33±0.333kn 3.00±0.000ghi 12.5 mg mL-1 0.00±0.00o 1.00±0.577no 2.33±0.333ijk 4.67±0.333ef 25 mg mL-1 0.00±0.00o 2.67±0.333hij 5.00±0.000e 7.67±0.333c 50 mg mL-1 0.00±0.00o 4.00±0.577fg 7.33±0.333c 10.00±0.000a Areca catechu 1.56 mg mL-1 0.00±0.00m 0.00±0.00m 0.00±0.00m 1.33±0.333jkl 3.12 mg mL-1 0.00±0.00m 0.00±0.00m 0.33±0.333lm 1.67±0.333jk 6.25 mg mL-1 0.00±0.00m 0.00±0.00m 1.67±0.333jk 3.00±0.577hi 12.5 mg mL-1 0.00±0.00m 0.67±0.333klm 2.33±0.333ij 5.67±0.667ef 25 mg mL-1 0.00±0.00m 2.00±0.577ij 5.00±1.155fg 7.67±0.882cd 50 mg mL-1 0.00±0.00m 4.00±0.577gh 7.33±0.333cd 9.00±0.577ab Amomum subulatum 1.56 mg mL-1 0.00±0.00m 0.00±0.000m 0.33±0.333lm 1.00±0.000 klm 3.12 mg mL-1 0.00±0.00m 0.00±0.000m 1.00±0.000klm 1.67±0.000ijk 6.25 mg mL-1 0.00±0.00m 0.00±0.000m 1.33±0.333jkl 2.33±0.333hij 12.5 mg mL-1 0.00±0.00m 0.00±0.000m 1.67±0.333ijk 3.33±0.333h 25 mg mL-1 0.00±0.00m 1.67±0.333ijk 3.00±0.577h 6.00±0.577def 50 mg mL-1 0.00±0.00m 2.67±0.882hi 5.67±0.333efg 7.33±0.667bc Vitex negundo 1.56 mg mL-1 0.00±0.00o 0.00±0.00o 0.00±0.000k 0.67±0.667mno 3.12 mg mL-1 0.00±0.00o 0.00±0.00o 0.33±0.333ijk 1.00±0.577lo 6.25 mg mL-1 0.00±0.00o 0.00±0.00o 0.67±0.333hk 3.00±0.577fi 12.5 mg mL-1 0.00±0.00o 0.00±0.00o 1.67±0.333gj 4.00±0.577ef 25 mg mL-1 0.00±0.00o 1.00±0.577lo 2.33±0.333eh 4.33±0.333de 50 mg mL-1 0.00±0.00o 2.00±0.577il 4.00±0.577cde 7.00±0.577c Acacia nilotica leaves 1.56 mg mL-1 0.00±0.00o 0.00±0.00o 0.00±0.00o 0.33±0.333no 3.12 mg mL-1 0.00±0.00o 0.00±0.00o 0.00±0.00o 1.33±0.333klm 6.25 mg mL-1 0.00±0.00o 0.00±0.00o 0.33±0.333no 1.67±0.333jkl 12.5 mg mL-1 0.00±0.00o 0.00±0.00o 0.67±0.333mno 2.33±0.333ij 25 mg mL-1 0.00±0.00o 0.67±0.333mno 2.00±0.000ijk 3.33±0.333gh 50 mg mL-1 0.00±0.00o 1.67±0.333jkl 3.33±0.333gh 6.00±0.577d Arundo donax 1.56 mg mL-1 0.00±0.00l 0.00±0.00l 0.00±0.00l 0.33±0.333kl 3.12 mg mL-1 0.00±0.00l 0.00±0.00l 0.00±0.00l 0.67±0.333ikl 6.25 mg mL-1 0.00±0.00l 0.00±0.00l 0.33±0.333kl 1.33±0.333ij 12.5 mg mL-1 0.00±0.00l 0.00±0.00l 1.00±0.000ijk 2.67±0.333fg 25 mg mL-1 0.00±0.00l 0.00±0.00l 2.33±0.333gh 4.33±0.333e 50 mg mL-1 0.00±0.00l 0.67±0.333jkl 3.33±0.333f 5.67±0.333c Note: Observations on the motility of worms were recorded on 0 hr pre-exposure, and 2, 4, 6, 8, 10, 12 and 24 hr post-exposure. However, the data of 0, 2, 6 and 10 hr has been shown in the table as it was deemed enough to meet the objectives of the bioassay.

39 Table 17: In vitro effect of different fractions of crude aqueous methanol extracts of Ferula assa-foetida on survival of H. contortus of sheep in comparison with Levamisole

Treatments Number of dead worms (Mean±SE) at different hours Hours post-exposure 0 hr 2 hr 6 hr 10 hr Levamisole 0.5 mg mL-1 0.00±0.00p 10.00±0.000a 10.00±0.000a 10.00±0.000a PBS 0.00±0.00p 0.00±0.00p 0.00±0.00p 0.00±0.00p Chloroform 1.56 mg mL-1 0.00±0.00p 0.00±0.00p 1.00±0.577nop 2.67±0.333jm 3.12 mg mL-1 0.00±0.00p 0.00±0.00p 2.00±0.577lmn 3.67±0.333hij 6.25 mg mL-1 0.00±0.00p 0.67±0.333op 2.33±0.333klm 5.00±0.577efg 12.5 mg mL-1 0.00±0.00p 1.67±0.333mno 4.00±0.577ghi 7.00±0.577c 25 mg mL-1 0.00±0.00p 2.33±0.333klm 5.00±0.577efg 9.67±0.333a 50 mg mL-1 0.00±0.00p 4.33±0.882fgh 7.00±0.577c 10.00±0.000a Ethyle acetate 1.56 mg mL-1 0.00±0.00m 0.00±0.00m 1.67±0.333jk 4.00±0.577fg 3.12 mg mL-1 0.00±0.00m 0.33±0.333lm 2.33±0.333ij 5.33±0.333d 6.25 mg mL-1 0.00±0.00m 1.33±0.333jk 3.67±0.667fgh 8.00±0.577b 12.5 mg mL-1 0.00±0.00m 1.67±0.333jk 5.00±0.577de 10.00±0.000a 25 mg mL-1 0.00±0.00m 2.33±0.333ij 6.67±0.333c 10.00±0.000a 50 mg mL-1 0.00±0.00m 3.33±0.333gh 8.00±0.577b 10.00±0.000a Petroleum spirit 1.56 mg mL-1 0.00±0.00m 0.00±0.00m 0.00±0.00m 1.67±0.333hk 3.12 mg mL-1 0.00±0.00m 0.00±0.00m 0.67±0.333klm 2.00±0.577gj 6.25 mg mL-1 0.00±0.00m 0.33±0.333lm 1.67±0.333hk 2.67±0.333fgh 12.5 mg mL-1 0.00±0.00m 1.00±0.577jm 2.33±0.882fi 4.33±0.333cd 25 mg mL-1 0.00±0.00m 1.33±0.333il 3.00±0.577fg 4.67±0.882cd 50 mg mL-1 0.00±0.00m 2.33±0.667fi 4.67±0.333cd 6.33±0.333b Aqueous 1.56 mg mL-1 0.00±0.00m 0.00±0.00m 0.00±0.00m 1.00±0.000jm 3.12 mg mL-1 0.00±0.00m 0.00±0.00m 0.33±0.333lm 1.67±0.333hk 6.25 mg mL-1 0.00±0.00m 0.00±0.00m 0.67±0.333klm 2.33±0.333ghi 12.5 mg mL-1 0.00±0.00m 0.33±0.333lm 1.33±0.333il 2.67±0.333fgh 25 mg mL-1 0.00±0.00m 1.00±0.000jm 2.00±0.577hij 4.33±0.882cde 50 mg mL-1 0.00±0.00m 1.33±0.333il 3.67±0.667def 5.33±0.882bc Note: Observations on the motility of worms were recorded on 0 hr pre-exposure, and 2, 4, 6, 8, 10, 12 and 24 hr post-exposure. However, the data of 0, 2, 6 and 10 hr has been shown in the table as it was deemed enough to meet the objectives of the bioassay.

40 Table 18: In vitro effect of different fractions of crude aqueous methanol extracts of Acacia nilotica bark on survival of H. contortus of sheep in comparison with Levamisole

Treatments Number of dead worms (Mean±SE) at different hours Hours post-exposure 0 hr 2 hr 6 hr 10 hr Levamisole 0.5 mg mL-1 0.00±0.00k 10.00±0.00a 10.00±0.00a 10.00±0.00a PBS 0.00±0.00k 0.00±0.00k 0.00±0.00k 0.00±0.00k Chloroform 1.56 mg mL-1 0.00±0.00k 0.00±0.00k 0.00±0.00k 1.00±0.000hk 3.12 mg mL-1 0.00±0.00k 0.00±0.00k 0.33±0.333jk 1.33±0.333gj 6.25 mg mL-1 0.00±0.00k 0.00±0.00k 0.67±0.333ijk 1.67±0.333ghi 12.5 mg mL-1 0.00±0.00k 0.00±0.00k 1.00±0.577hk 3.00±1.000ef 25 mg mL-1 0.00±0.00k 0.67±0.333ijk 2.00±0.000fgh 4.00±0.577de 50 mg mL-1 0.00±0.00k 2.00±0.000fgh 4.00±0.577de 6.67±0.882bc Ethyle acetate 1.56 mg mL-1 0.00±0.00n 0.00±0.00n 0.33±0.333mn 1.00±0.000kn 3.12 mg mL-1 0.00±0.00n 0.00±0.00n 0.67±0.333lmn 1.67±0.333il 6.25 mg mL-1 0.00±0.00n 0.00±0.00n 1.00±0.000kn 2.33±0.667g-j 12.5 mg mL-1 0.00±0.00n 0.67±0.333lmn 2.33±0.333gj 4.00±0.577ef 25 mg mL-1 0.00±0.00n 1.67±0.333ijkl 2.67±0.333ghi 5.00±0.577de 50 mg mL-1 0.00±0.00n 2.67±0.667ghi 5.00±0.577de 8.33±0.882bc Petroleum spirit 1.56 mg mL-1 0.00±0.00h 0.00±0.00h 0.00±0.00h 0.33±0.333gh 3.12 mg mL-1 0.00±0.00h 0.00±0.00h 0.00±0.00h 0.67±0.333fgh 6.25 mg mL-1 0.00±0.00h 0.00±0.00h 0.33±0.333gh 1.00±0.000efg 12.5 mg mL-1 0.00±0.00h 0.00±0.00h 0.67±0.333fgh 1.33±0.333ef 25 mg mL-1 0.00±0.00h 0.00±0.00h 1.00±0.000efg 1.67±0.333de 50 mg mL-1 0.00±0.00h 0.00±0.333gh 1.67±0.333de 4.00±0.577b Aqueous 1.56 mg mL-1 0.00±0.00i 0.00±0.00i 0.00±0.00i 0.00±0.00i 3.12 mg mL-1 0.00±0.00i 0.00±0.00i 0.00±0.00i 0.33±0.333i 6.25 mg mL-1 0.00±0.00i 0.00±0.00i 0.00±0.00i 1.00±0.000gh 12.5 mg mL-1 0.00±0.00i 0.00±0.00i 0.00±0.00i 1.33±0.333fg 25 mg mL-1 0.00±0.00i 0.00±0.00i 1.33±0.333fg 2.33±0.333cd 50 mg mL-1 0.00±0.00i 0.00±0.00i 2.00±0.577de 3.33±0.333b Note: Observations on the motility of worms were recorded on 0 hr pre-exposure, and 2, 4, 6, 8, 10, 12 and 24 hr post-exposure. However, the data of 0, 2, 6 and 10 hr has been shown in the table as it was deemed enough to meet the objectives of the bioassay.

41 Table 19: In vitro effect of different fractions of crude aqueous methanol extracts of Areca catechu on survival of H. contortus of sheep in comparison with Levamisole

Treatments Number of dead worms (Mean±SE) at different hours Hours post-exposure 0 hr 2 hr 6 hr 10 hr Levamisole 0.5 mg mL-1 0.00±0.00 m 10.00±0.00a 10.00±0.00a 10.00±0.00a PBS 0.00±0.00m 0.00±0.00m 0.00±0.00m 0.00±0.00m Chloroform 1.56 mg mL-1 0.00±0.00m 0.00±0.00m 0.00±0.00m 0.67±0.333klm 3.12 mg mL-1 0.00±0.00m 0.00±0.00m 0.67±0.667klm 1.67±0.333hk 6.25 mg mL-1 0.00±0.00m 0.00±0.00m 1.00±0.000jm 2.33±0.333fi 12.5 mg mL-1 0.00±0.00m 0.00±0.00m 2.33±0.333fi 3.00±0.000efg 25 mg mL-1 0.00±0.00m 0.00±0.00m 2.67±0.667eh 4.33±0.333cd 50 mg mL-1 0.00±0.00m 0.33±0.333lm 3.33±0.882ef 5.33±0.667bc Ethyle acetate 1.56 mg mL-1 0.00±0.00o 0.00±0.00o 0.00±0.00o 1.00±0.000lo 3.12 mg mL-1 0.00±0.00o 0.00±0.00o 0.67±0.333mno 1.67±0.333klm 6.25 mg mL-1 0.00±0.00o 0.00±0.00o 1.33±0.333kn 2.33±0.333ijk 12.5 mg mL-1 0.00±0.00o 0.33±0.333no 2.33±0.333ijk 3.67±0.333fgh 25 mg mL-1 0.00±0.00o 0.67±0.333mno 3.67±0.667fgh 5.00±0.577de 50 mg mL-1 0.00±0.00o 1.67±0.882klm 4.67±1.202def 6.67±0.882bc Petroleum spirit 1.56 mg mL-1 0.00±0.00j 0.00±0.00j 0.00±0.00j 0.00±0.00j 3.12 mg mL-1 0.00±0.00j 0.00±0.00j 0.00±0.00j 0.33±0.333ij 6.25 mg mL-1 0.00±0.00j 0.00±0.00j 0.00±0.00j 0.67±0.333hij 12.5 mg mL-1 0.00±0.00j 0.00±0.00j 0.33±0.333ij 1.33±0.333fgh 25 mg mL-1 0.00±0.00j 0.00±0.00j 0.67±0.333hij 2.00±0.000e 50 mg mL-1 0.00±0.00j 0.00±0.00j 2.00±0.577ef 3.00±0.577c Aqueous 1.56 mg mL-1 0.00±0.00m 0.00±0.00m 0.00±0.00m 0.33±0.333lm 3.12 mg mL-1 0.00±0.00m 0.00±0.00m 0.33±0.333lm 1.00±0.000jkl 6.25 mg mL-1 0.00±0.00m 0.00±0.00m 1.00±0.000jkl 1.67±0.333hij 12.5 mg mL-1 0.00±0.00m 0.00±0.00m 1.67±0.333hij 2.67±0.333efg 25 mg mL-1 0.00±0.00m 0.67±0.333klm 2.00±0.000ghi 3.00±0.577ef 50 mg mL-1 0.00±0.00m 1.33±0.333ijk 2.67±0.333efg 4.33±0.667bc Note: Observations on the motility of worms were recorded on 0 hr pre-exposure, and 2, 4, 6, 8, 10, 12 and 24 hr post-exposure. However, the data of 0, 2, 6 and 10 hr has been shown in the table as it was deemed enough to meet the objectives of the bioassay.

42 Table 20: In vitro effect of different fractions of crude aqueous methanol extracts of Amomum subulatum on survival of H. contortus of sheep in comparison with Levamisole

Treatments Number of dead worms (Mean±SE) at different hours Hours post-exposure 0 hr 2 hr 6 hr 10 hr Levamisole 0.5 mg mL-1 0.00±0.00r 10.00±0.00a 10.00±0.00a 10.00±0.00a PBS 0.00±0.00r 0.00±0.00r 0.00±0.00r 0.00±0.00r Chloroform 1.56 mg mL-1 0.00±0.00r 0.00±0.00r 0.67±0.667qr 1.67±0.333nq 3.12 mg mL-1 0.00±0.00r 0.00±0.00r 1.33±0.333or 2.67±0.333ko 6.25 mg mL-1 0.00±0.00r 0.67±0.333qr 2.00±0.000mq 3.00±0.000jn 12.5 mg mL-1 0.00±0.00r 1.33±0.333or 2.67±0.667ko 4.00±0.577gk 25 mg mL-1 0.00±0.00r 1.67±0.333nq 4.33±0.667fj 5.33±0.667efg 50 mg mL-1 0.00±0.00r 3.33±0.882im 5.67±0.882ef 7.67±0.333bc Ethyle acetate 1.56 mg mL-1 0.00±0.00r 0.00±0.00r 1.33±0.333opq 2.67±0.333kn 3.12 mg mL-1 0.00±0.00r 0.67±0.333qr 2.33±0.333lo 3.33±0.667jkl 6.25 mg mL-1 0.00±0.00r 1.00±0.00pqr 2.67±0.333kn 4.33±0.882hij 12.5 mg mL-1 0.00±0.00r 1.67±0.333nq 4.33±0.333hij 6.00±0.577ef 25 mg mL-1 0.00±0.00r 2.00±0.577mp 5.33±0.333fgh 9.00±0.577ab 50 mg mL-1 0.00±0.00r 4.33±0.333hij 7.00±0.577de 10.00±0.000a Petroleum spirit 1.56 mg mL-1 0.00±0.00n 0.00±0.00n 0.00±0.00n 1.00±0.000klm 3.12 mg mL-1 0.00±0.00n 0.00±0.00n 0.00±0.00n 1.67±0.333ijk 6.25 mg mL-1 0.00±0.00n 0.00±0.00n 0.67±0.333lmn 2.33±0.333ghi 12.5 mg mL-1 0.00±0.00n 0.00±0.00n 1.33±0.333jkl 2.67±0.333fgh 25 mg mL-1 0.00±0.00n 0.00±0.00n 2.00±0.577hij 3.33±0.333def 50 mg mL-1 0.00±0.00n 0.00±0.00n 3.00±0.577efg 4.67±0.667bc Aqueous 1.56 mg mL-1 0.00±0.00k 0.00±0.00k 0.00±0.00k 0.33±0.333jk 3.12 mg mL-1 0.00±0.00k 0.00±0.00k 0.00±0.00k 0.67±0.333ijk 6.25 mg mL-1 0.00±0.00k 0.00±0.00k 0.00±0.00k 1.33±0.333ghi 12.5 mg mL-1 0.00±0.00k 0.00±0.00k 0.00±0.00k 2.33±0.333ef 25 mg mL-1 0.00±0.00k 0.00±0.00k 1.33±0.333ghi 2.67±0.333de 50 mg mL-1 0.00±0.00k 0.00±0.00k 2.33±0.333ef 5.00±0.577b Note: Observations on the motility of worms were recorded on 0 hr pre-exposure, and 2, 4, 6, 8, 10, 12 and 24 hr post-exposure. However, the data of 0, 2, 6 and 10 hr has been shown in the table as it was deemed enough to meet the objectives of the bioassay.

43 Table 21: In vitro effect of different fractions of crude aqueous methanol extracts of Vitex negundo on survival of H. contortus of sheep in comparison with Levamisole

Treatments Number of dead worms (Mean±SE) at different hours Hours post-exposure 0 hr 2 hr 6 hr 10 hr Levamisole 0.5 mg mL-1 0.00±0.00k 10.00±0.00a 10.00±0.00a 10.00±0.00a PBS 0.00±0.00k 0.00±0.00k 0.00±0.00k 0.00±0.00k Chloroform 1.56 mg mL-1 0.00±0.00k 0.00±0.00k 0.00±0.000k 0.33±0.333jk 3.12 mg mL-1 0.00±0.00k 0.00±0.00k 0.67±0.333ijk 1.33±0.333gj 6.25 mg mL-1 0.00±0.00k 0.00±0.00k 1.00±0.000hk 1.67±0.333fi 12.5 mg mL-1 0.00±0.00k 0.00±0.00k 1.33±0.333gj 2.33±0.333dg 25 mg mL-1 0.00±0.00k 0.33±0333jk 2.00±0.577eh 3.00±0.577cde 50 mg mL-1 0.00±0.00k 1.00±0.577hk 3.00±0.577cde 4.67±0.667b Ethyle acetate 1.56 mg mL-1 0.00±0.00 m 0.00±0.00 m 0.00±0.000m 0.33±0.333lm 3.12 mg mL-1 0.00±0.00m 0.00±0.00m 0.67±0.333klm 1.33±0.333ijk 6.25 mg mL-1 0.00±0.00 m 0.00±0.00 m 1.33±0.333ijk 2.00±0.000ghi 12.5 mg mL-1 0.00±0.00m 0.00±0.00m 1.67±0.333hij 2.33±0.333gh 25 mg mL-1 0.00±0.00 m 0.67±0.333klm 2.00±0.000 ghi 4.00±0.577de 50 mg mL-1 0.00±0.00m 1.67±0.333hij 3.33±0.882 ef 6.00±0.577b Petroleum spirit 1.56 mg mL-1 0.00±0.00k 0.00±0.00k 0.00±0.00k 0.33±0.333jk 3.12 mg mL-1 0.00±0.00k 0.00±0.00k 0.00±0.00k 0.67±0.333ijk 6.25 mg mL-1 0.00±0.00k 0.00±0.00k 0.00±0.00k 1.33±0.333ghi 12.5 mg mL-1 0.00±0.00k 0.00±0.00k 0.67±0.333ijk 2.00±0.577efg 25 mg mL-1 0.00±0.00k 0.00±0.00k 1.33±0.333ghi 2.67±0.333de 50 mg mL-1 0.00±0.00k 0.00±0.00k 2.67±0.667de 4.33±0.333b Aqueous 1.56 mg mL-1 0.00±0.00l 0.00±0.00l 0.00±0.00l 0.33±0.333kl 3.12 mg mL-1 0.00±0.00l 0.00±0.00l 0.00±0.00l 1.33±0.333ijk 6.25 mg mL-1 0.00±0.00l 0.00±0.00l 0.67±0.333jkl 1.67±0.333hij 12.5 mg mL-1 0.00±0.00l 0.00±0.00l 1.00±0.577ijkl 2.67±0.667fgh 25 mg mL-1 0.00±0.00l 0.33±0.333kl 1.67±0.333hij 3.67±0.882ef 50 mg mL-1 0.00±0.00l 0.67±0.667jkl 3.00±0.577fg 5.67±0.667bc Note: Observations on the motility of worms were recorded on 0 hr pre-exposure, and 2, 4, 6, 8, 10, 12 and 24 hr post-exposure. However, the data of 0, 2, 6 and 10 hr has been shown in the table as it was deemed enough to meet the objectives of the bioassay.

44 Table 22: In vitro effect of different fractions of crude aqueous methanol extracts of Acacia nilotica leaves on survival of H. contortus of sheep in comparison with Levamisole

Treatments Number of dead worms (Mean±SE) at different hours Hours post-exposure 0 hr 2 hr 6 hr 10 hr Levamisole 0.5 mg mL-1 0.00±0.00j 10.00±0.00a 10.00±0.00a 10.00±0.00a PBS 0.00±0.00j 0.00±0.00j 0.00±0.00j 0.00±0.00j Chloroform 1.56 mg mL-1 0.00±0.00j 0.00±0.00j 0.00±0.00j 0.67±0.333hij 3.12 mg mL-1 0.00±0.00j 0.00±0.00j 0.33±0.333ij 1.33±0.333fi 6.25 mg mL-1 0.00±0.00j 0.00±0.00j 1.00±0.000gj 2.00±0.000efg 12.5 mg mL-1 0.00±0.00j 0.33±0.333ij 2.00±0.577efg 3.33±0.667cd 25 mg mL-1 0.00±0.00j 1.00±0.577gj 2.33±0.333ef 4.00±0.577c 50 mg mL-1 0.00±0.00j 2.00±0.577efg 4.00±0.577c 5.33±0.333a Ethyle acetate 1.56 mg mL-1 0.00±0.00m 0.00±0.00m 0.67±0.333klm 1.67±0.333ijk 3.12 mg mL-1 0.00±0.00m 0.00±0.00m 1.33±0.333ijk 2.00±0.000hij 6.25 mg mL-1 0.00±0.00m 0.33±0.333lm 1.67±0.333ijk 3.33±0.333fg 12.5 mg mL-1 0.00±0.00m 0.67±0.333klm 2.67±0.333gh 4.00±0.577ef 25 mg mL-1 0.00±0.00m 1.00±0.577jkl 4.00±0.577ef 6.67±0.333c 50 mg mL-1 0.00±0.00m 2.00±0.577hij 5.67±0.333d 9.67±0.333a Petroleum spirit 1.56 mg mL-1 0.00±0.00g 0.00±0.00g 0.00±0.00g 0.00±0.000g 3.12 mg mL-1 0.00±0.00g 0.00±0.00g 0.00±0.00g 0.33±0.333fg 6.25 mg mL-1 0.00±0.00g 0.00±0.00g 0.33±0.333fg 1.33±0.333ef 12.5 mg mL-1 0.00±0.00g 0.00±0.00g 0.67±0.333fg 1.33±0.333ef 25 mg mL-1 0.00±0.00g 0.67±0.333fg 2.33±0.333d 3.00±0.577d 50 mg mL-1 0.00±0.00g 1.00±0.577efg 3.00±0.577d 4.00±0.577c Aqueous 1.56 mg mL-1 0.00±0.00i 0.00±0.00i 0.00±0.00i 0.67±0.333 ghi 3.12 mg mL-1 0.00±0.00i 0.00±0.00i 0.33±0.333hi 1.00±0.000 f-i 6.25 mg mL-1 0.00±0.00i 0.00±0.00i 0.67±0.667ghi 1.33±0.333 e-h 12.5 mg mL-1 0.00±0.00i 0.00±0.00i 1.00±0.000fi 1.67±0.333 efg 25 mg mL-1 0.00±0.00i 0.33±0.333hi 1.67±0.333efg 2.33±0.667 de 50 mg mL-1 0.00±0.00i 0.67±0.333ghi 3.00±0.577cd 3.67±0.333 bc Note: Observations on the motility of worms were recorded on 0 hr pre-exposure, and 2, 4, 6, 8, 10, 12 and 24 hr post-exposure. However, the data of 0, 2, 6 and 10 hr has been shown in the table as it was deemed enough to meet the objectives of the bioassay.

45 Table 23: In vitro effect of different fractions of crude aqueous methanol extracts of Arundo donax on survival of H. contortus of sheep in comparison with Levamisole

Treatments Number of dead worms (Mean±SE) at different hours Hours post-exposure 0 hr 2 hr 6 hr 10 hr Levamisole 0.5 mg mL-1 0.00±0.00o 10.00±0.00a 10.00±0.00a 10.00±0.00a PBS 0.00±0.00o 0.00±0.00o 0.00±0.00o 0.00±0.00o Chloroform 1.56 mg mL-1 0.00±0.00o 0.00±0.00o 0.00±0.00o 1.00±0.000l-o 3.12 mg mL-1 0.00±0.00o 0.00±0.00o 0.67±0.333mno 1.33±0.333kn 6.25 mg mL-1 0.00±0.00o 0.33±0.333no 1.33±0.333kn 2.33±0.333hk 12.5 mg mL-1 0.00±0.00o 0.67±0.667mno 2.33±0.333hk 3.67±0.333dg 25 mg mL-1 0.00±0.00o 1.33±0.333kn 3.00±0.000fi 4.33±0.667cde 50 mg mL-1 0.00±0.00o 2.33±0.667hk 4.67±0.333cd 6.67±0.667b Ethyle acetate 1.56 mg mL-1 0.00±0.00o 0.00±0.00o 0.00±0.00o 0.67±0.333mno 3.12 mg mL-1 0.00±0.00o 0.00±0.00o 0.00±0.00o 1.33±0.333lo 6.25 mg mL-1 0.00±0.00o 0.00±0.00o 1.00±0.577mno 2.67±0.333hk 12.5 mg mL-1 0.00±0.00o 0.67±0.333mno 2.33±0.333il 3.33±0.667fi 25 mg mL-1 0.00±0.00o 1.00±0.577mno 3.33±0.882fi 4.33±0.667ef 50 mg mL-1 0.00±0.00o 3.00±1.155gj 5.00±0.57de 7.00±0.577bc Petroleum spirit 1.56 mg mL-1 0.00±0.00l 0.00±0.00l 0.00±0.00l 0.67±0.333jkl 3.12 mg mL-1 0.00±0.00l 0.00±0.00l 0.00±0.00l 1.00±0.000ijk 6.25 mg mL-1 0.00±0.00l 0.00±0.00l 0.33±0.333kl 1.33±0.333hij 12.5 mg mL-1 0.00±0.00l 0.00±0.00l 0.67±0.333jkl 1.67±0.333ghi 25 mg mL-1 0.00±0.00l 0.67±0.667 jkl 1.33±0.333hij 2.33±0.333efg 50 mg mL-1 0.00±0.00l 1.00±0.577 ijk 2.33±0.333efg 4.00±0.577bc Aqueous 1.56 mg mL-1 0.00±0.00l 0.00±0.00l 0.00±0.00l 0.33±0.333kl 3.12 mg mL-1 0.00±0.00l 0.00±0.00l 0.00±0.00l 1.33±0.667hij 6.25 mg mL-1 0.00±0.00l 0.00±0.00l 0.00±0.00l 1.67±0.333ghi 12.5 mg mL-1 0.00±0.00l 0.00±0.00l 0.67±0.333jkl 2.33±0.333efg 25 mg mL-1 0.00±0.00l 0.00±0.00l 1.33±0.333hij 3.00±0.577de 50 mg mL-1 0.00±0.00l 0.00±0.00l 2.33±0.667efg 5.00±0.577b Note: Observations on the motility of worms were recorded on 0 hr pre-exposure, and 2, 4, 6, 8, 10, 12 and 24 hr post-exposure. However, the data of 0, 2, 6 and 10 hr has been shown in the table as it was deemed enough to meet the objectives of the bioassay.

Table 24: Ranking of efficacy of different fractions of plants against H. contortus

Plant Ranking of Efficacy of Different Fractions of Plants 1st 2nd 3rd 4th Ferula assa-foetida Ethyle acetate Chloroform Petroleum spirit Aqueous Acacia nilotica bark Ethyle acetate Chloroform Petroleum spirit Aqueous Areca catechue Ethyle acetate Chloroform Aqueous Petroleum spirit Amomum subulatum Ethyle acetate Chloroform Aqueous Petroleum spirit Vitex negundo Ethyle acetate Aqueous Aqueous Petroleum spirit Acacia nilotica leaves Ethyle acetate Chloroform Petroleum spirit Aqueous Arundo donax Ethyle acetate Chloroform Aqueous Petroleum spirit

46 4.2.2. Egg hatch assay Crude aqueous methanol extract of Amomum subulatum was found to be the most effective ovicidal among all the plants included in the study based on its lowest LC50 (13.1872 µg mL-1) followed by Ferula assa-foetida, Areca catechu, Vitex negundo, Arundo donax, Acacia - nilotica bark and Acacia nilotica leaves (Table 25). Indeed, albendazole (LC50 = 0.0345 µg mL 1) used as reference drug, was the most effective. Dose and time dependent ovicidal effects were recorded for all the plants. The ovicidal effects were more pronounced in the eggs exposed to chloroform fractions of Amomum subulatum, Ferula assa-foetida , Arundo donax and Acacia nilotica bark; aqueous fractions of Areca catechu and Vitex negundo; and ethyle acetate fraction of Acacia nilotica leaves (Table 25) followed, in general, by ethyle acetate fractions. Aqueous and petroleum fractions generally (except Arecha catechue and Arundo donax) had low ovicidal efficacy. It is evident from Table 26 that majority (08/10) of the top 10 highly ovicidal extracts/fractions were of those of Amomum subulatum and Ferula assa-foetida.

Table 25: Percent egg hatch and LC50 of crude aqueous methanol extracts and fractions of different plants

-1 Plant CAME/fraction concentrations µg mL LC50 µg mL-1

1.2 12 120 1200 12000 Per cent Egg Hatch Albendazole 79 64 40 5 0 0.0345 Crude aqueous methanol extracts Amomum subulatum 41 39 28 19 6 13.1872 Ferula assa-foetida 81 52 30 7 0 16.9126 Areca catechu 85 63 44 25 2 45.5659 Vitex negundo 77 66 46 31 12 65.7405 Arundo donax 80 75 54 40 19 200.1246 Acacia nilotica Bark 80 72 58 35 22 201.0032 Acacia nilotica leaves 81 76 68 40 35 769.2485 Fractions Amomum subulatum Choloroform fraction 37 36 32 26 12 18.1413 Ethyl acetate fraction 44 36 27 16 3 18.6102 Aqueous fraction 43 41 32 22 16 20.4178 Petroleum spirit fraction 44 36 32 26 12 253.9106 Ferula assa-foetida Choloroform fraction 68 45 23 6 0 6.9698 Ethyl acetate fraction 71 51 25 0 0 10.6419 Aqueous fraction 77 59 41 19 5 29.6247 Petroleum spirit fraction 82 67 53 45 32 381.05 Areca catechu Aqueous fraction 82 68 39 18 3 38.9270 Ethyl acetate fraction 87 65 38 20 3 45.7080 Chloroform fraction. 83 71 49 35 4 74.2700 Petroleum spirit fraction 88 75 52 37 12 163.5530

47 Table 25 Contd. -1 Plant CAME/fraction concentrations µg mL LC50 µg mL-1

1.2 12 120 1200 12000 Per cent Egg Hatch Vitex negundo Aqueous fraction 72 60 39 33 13 38.7527 Petroleum spirit fraction 74 57 49 35 17 61.5036 Ethyl acetate fraction 76 65 49 38 16 92.3089 Choloroform fraction 88 65 48 35 25 191.0117 Arundo donax Choloroform fraction 83 63 48 36 12 91.0189 Aqueous fraction 76 68 59 42 19 174.8342 Ethyl acetate fraction 81 77 56 44 23 319.8749 Petroleum spirit fraction 84 70 57 45 31 467.5344 Acacia nilotica bark Chloroform fraction 82 49 44 29 16 50.9734 Ethyl acetate fraction 81 73 37 26 5 52.5873 Aqueous fraction 82 76 55 34 23 222.8358 Petroleum spirit fraction. 84 70 54 39 28 289.2595 Acacia nilotica leaves Ethyl acetate fraction 80 68 55 33 18 129.4961 Choloroform fraction 83 69 48 34 23 156.5536 Aqueous fraction 85 79 68 46 34 1046.5714 Petroleum spirit fraction 83 78 70 59 47 8408.9702

Table 26: Top 10 highly ovicidal plant crude aqueous methanol extracts and/or fractions in comparison with control

-1 Plant CAME/fraction concentrations µg mL LC50 µg mL-1

1.2 12 120 1200 12000 Per cent Egg Hatch Albendazole 79 64 40 5 0 0.0345 1. Ferula assa-foetida choloroform fraction 68 45 23 6 0 6.9698 2. Ferula assa-foetida ethyl acetate fraction 71 51 25 0 0 10.6419 3. Ammoum subulatum CAME 41 39 28 19 6 13.1872 4. Ferula assa-foetida CAME 81 52 30 7 0 16.9126 5. Amomum subulatum choloroform fraction 37 36 32 26 12 18.1413 6. Amomum subulatum ethyl acetate fraction 44 36 27 16 3 18.6102 7. Amomum subulatum aqueous fraction 43 41 32 22 16 20.4178 8. Ferula assa-foetida aqueous fraction 77 59 41 19 5 29.6247 9. Vitex negundo aqueous fraction 72 60 39 33 13 38.7527 10. Areca catechu aqueous fraction 82 68 39 18 3 38.9270

4.2.3. Regression values and correlation of regression of the effect of different plants on egg hatching The data of correlation of regression (Table 27) indicate that the best dose-dependant effect was found with crude aqueous methanol extract of Ferula assa-foetida followed by Vitex negundo, Acacia nilotica bark, Amomum subulatum, Arundo donax, Areca catechú and Acacia nilotica leaves. The dose-dependant effects were more pronounced with petroleum spirit fractions of Vitex negundo, Acacia nilotica bark, Amomum subulatum, Arundo donax, and Areca catechú; aqueous fractions of Ferula assa-foetida; and ethyle acetate fraction of Acacia nilotica leaves (Table 27). The top 10 plants (Table 28) having most linear (i.e., dose dependant) effect

48 were Acacia nilotica leaves (petroleum spirit, ethyle acetate and chloroform fractions); Acacia nilotica bark (petroleum spirit and CAME); Ferula assa-foetida (CAME and aqueous fraction); Arundo donax and Amomum subulatum (petroleum spirit fraction), and Vitex negundo (CAME). It is evident from Table 28 that of the top 10 CAME and/or fractions having most linear effect, 05 pertained to Acacia nilotica, 02 pertained to Ferula assa-foetida and 01 each to Arundo donax, Amomum subulatum and Vitex negundo. The top 10 treatments having most linear (i.e., dose dependant) effect included 04 petroleum spirit, 03 CAME and 01 each ethyle acetate, chloroform and aqueous fractions (Table 28). Table 27: Regression values and correlation of regression of the effect of different plants on egg hatching Plant CAME/Fraction Regression values and correlation of regression Crude aqueous methanol extract Ferula assa-foetida y = -0.7636x + 8.2287, R2 = 0.9919 Vitex negundo y = -0.4737x + 7.2822 ,R2 = 0.9856 Acacia nilotica bark y = -0.4196x + 7.2252, R2 = 0.9838 Amomum subulatum y = -0.5174x + 7.1318, R2 = 0.9806 Arundo donax y = -0.4367x + 7.3151, R2 = 0.9725 Areca catechú y = -0.7186x + 8.3477, R2 = 0.9522 Acacia nilotica leaves y = -0.3485x + 7.0513, R2 = 0.9358 Fractions Acacia nilotica leaves Ethyle acetate fraction y = -0.4414x + 7.2566, R2 = 0.9909 Petroleum spirit fraction y = -0.2603x + 6.8025, R2 = 0.9907 Chloroform fraction y = -0.4295x + 7.2311, R2 = 0.9902 Aqueous fraction y = -0.3804x + 7.2899,R2 = 0.9810 Acacia nilotica bark Petroleum spirit fraction y= -0.3957x + 7.161, R2 = 0.9935 Aqueous fraction y = -0.4427x + 7.3676, R2 = 0.9812 Ethyle acetate fraction y = -0.6302x + 7.9751, R2 = 0.9636 Chloroform fraction y = -0.4346x + 7.0458, R2 = 0.9332 Areca catechue Petroleum spirit fraction y = -0.4043x + 7.1851, R2 = 0.9852 Chloroform fraction y = -0.4243x + 6.8431, R2 = 0.9775 Aqueous fraction y = -0.5177x + 7.2313, R2 = 0.9750 Ethyle acetate fraction y = -1.2407x + 10.166, R2 = 0.7621 Vitex negundo Petroleum spirit fraction y = -0.3755x + 6.7982, R2 = 0.9765 Aqueous fraction y = -0.4111x + 6.8863, R2 = 0.9715 Ethyle acetate fraction y = -0.409x + 7.0308, R2 = 0.9740 Chloroform fraction y = -0.4468x + 7.3596, R2 = 0.9537 Arundo donax Petroleum spirit fraction y = -0.363x + 7.0581, R2 = 0.9940 Chloroform fraction y = -0.4948x + 7.4538, R2 = 0.9739 Ethyle acetate fraction y = -0.4124x + 7.2703, R2 = 0.9693 Aqueous fraction y = -0.383x + 7.0079, R2 = 0.9442 Amomum subulatum Petroleum spirit fraction y = -0.4043x + 7.1851, R2 = 0.9852 Chloroform fraction y = -0.4243x + 6.8431, R2 = 0.9775 Aqueous fraction y = -0.5177x + 7.2313, R2 = 0.9750 Ethyle acetate fraction y = -1.2407x + 10.166, R2 = 0.7621 Ferula assa-foetida Aqueous fraction y = -0.5873x + 7.6262, R2 = 0.9884 Petroleum spirit fraction y = -0.3332x + 6.8596, R2 = 0.9811 Ethyle acetate fraction y = -1.6131x + 11.174, R2 = 0.8567 Chloroform fraction y = -1.2349x + 9.8803, R2 = 0.8242

49 Table 28: Top 10 plant crude aqueous methanol extracts and/or fractions having best dose dependant ovicidal effects Plant CAME/Fraction Regression values and correlation of regression Arundo donax petroleum spirit fraction y = -0.363x + 7.0581, R2 = 0.9940 Acacia nilotica bark petroleum spirit fraction y= -0.3957x + 7.161, R2 = 0.9935 Ferula assa-foetida CAME y = -0.7636x + 8.2287, R2 = 0.9919 Acacia nilotica leaves ethyle acetate fraction y = -0.4414x + 7.2566, R2 = 0.9909 Acacia nilotica leaves petroleum spirit fraction y = -0.2603x + 6.8025, R2 = 0.9907 Acacia nilotica leaves chloroform fraction y = -0.4295x + 7.2311, R2 = 0.9902 Ferula assa-foetida aqueous fraction y = -0.5873x + 7.6262, R2 = 0.9884 Vitex negundo CAME y = -0.4737x + 7.2822 ,R2 = 0.9856 Amomum subulatum petroleum spirit fraction y = -0.4043x + 7.1851, R2 = 0.9852 Acacia nilotica bark CAME y = -0.4196x + 7.2252, R2 = 0.9838

4.3. In vivo anthelmintic activity Fecal egg count reduction in naturally parasitized sheep was the criterion for the evaluation of anthelmintic activity of different plants. Both crude powder and crude methanol extracts of all the plants included in this study exhibited dose dependant and varying levels of anthelmintic activity (Table 29). Efficacy ranking based on reduction in fecal egg counts as on day 12 post-treatment (PT) of crude powder and crude methanol extract of different plants has been presented in Table 30. Plants and doses used for evaluation in the current study are those commonly practiced in EVM in the study area. Likewise, crude powder and/or water decoctions of these plants are preferred by the traditional veterinary healers. Anthelmintic efficacy of crude powder of the plants included in this study ranged from 57.5 to 29.6%. The maximum reduction (57.5%) in fecal egg counts was recorded for Amomum subulatum @ 3 g kg-1 followed by Arundo donax @ 8 g kg-1, Ferula assa-foetida @ 1 g kg-1, Vitex negundo @ 8 g kg-1, Acacia nilotica leaves @ 8 g kg-1, Acacia nilotica bark @ 8 g kg-1 and Areca catechu @ 1 g kg-1. Anthelmintic efficacies of crude methanol extracts were not consistent with those recorded for crude powder. These were higher for majority of the plants and ranged from 87.6% (Ferula assa-foetida @ 1 g kg-1) to 31.5% (Arundo donax @ 8 g kg-1). 4.4. Salient findings on anthelmintic activity Comparative anthelmintic efficacy of crude aqueous methanol extracts/fractions of different plants in different tests employed in the current study has been presented in Table 30. Ranking of efficacy (Table 31) indicates that Ferula assa-foetida is effective in all the three tests used for evaluation of its anthelmintic activity; whereas, Acacia nilotica bark and Amomum subulatum were more effective against adult worms compared with their ovicidal activity. -1 Amomum subulatum, however, proved a better ovicidal (LC50=13.2 µg mL ) than Acacia -1 nilotica bark (LC50= 201.0 µg mL ). Likewise, Acacia nilotica leaves and Areca catechue were better against adult worms compared with their ovicidal effects. Chloroform and ethyle acetate

50 fractions appeared to have better anthelmintic effects against adult worms compared with their ovicidal activity. Like in vitro tests; Ferula assa-foetida, Acacia nilotica bark, Amomum subulatum, Acacia nilotica leaves and Areca catechue, were among the top five most effective plants in vivo studies. Crude aqueous methanol extracts (CAME) of plants proved superior in anthelmintic effects compared with crude powder (CP) in vivo studies. Table 29: Effect of crude powder and crude methanol extracts of different plants on eggs per gram of feces (Mean±SEM) in sheep naturally infected with mixed species of gastrointestinal nematodes in comparison with untreated and levamisole treated animals

Days Crude Powder Crude Aqueous Methanolic Extract Untreated Levamisole PT Ferula assa-foetida - - 0. 33 g kg-1 0.66 g kg-1 1g kg-1 0. 33 g kg- 0.66 g kg-1 1 g kg-1 0 g kg-1 7.5 mg kg-1 0 1558.3±72.3a 1550.0±78.5a 1516.7±86.3a 1516.7±86.3a 1483.3±82.3a 1475.0±88.3a 1575.0±69.2a 1575.0±69.2a 1400.0±51.6a 1341.7±58.3b 1225.0±70.4b 1316.7±60.1b 1158.3±47.3b 950.0±51.6b 1641.7±67.6a 58.3±15.4b 4 b (13.44) (19.23) (13.19) (21.91) (35.59) (-4.23) (96.30) (10.16) 1258.3±52.3b 1175.0±55.9b 991.7±98.7bc 1125.0±52.8c 566.7±27.9c 316.7±16.7c 1758.3±74.6a 8.3±8.3b 8 c c (34.61) (25.83) (61.79) (78.53) (-11.64) (99.47) (19.25) (24.19) 1183.3±60.1c 1100.0±50.0c 891.7±113.6c 908.3±53.9d 458.3±23.9c 183.3±16.7c 1825.0±79.3a 0.0±0.0b 12 (24.06) (29.03) (41.21) (40.11) (69.10) (87.57) (-15.87) (100.0) Areca catechu - - 0. 33 g kg-1 0.66 g kg-1 1 g kg-1 0. 33 g kg-1 0.66 g kg-1 1 g kg-1 0 g kg-1 7.5 mg kg-1 0 2458.3±76.8a 2433.3±83.3a 2391.7±92.6a 2383.3±91.9a 2375.0±89.2a 2350.0±85.7a 2466.7±79.2a 2475.0±85.4a 2325.0±72.7a 2275.0±82.4a 2116.7±85.3b 1716.7±65.4b 1483.3±55.8b 925.0±38.2b 2516.7±95.4a 66.7±10.5b 4 b b (11.50) (27.97) (37.55) (60.64) (-2.03) (97.31) (5.42) (6.51) 2225.0±61.6b 2133.3±71.5b 1891.7±82.1b 2616.7±118.8 1591.7±61.1b 1308.3±41.7b 625.0±21.4c 0.0±0.0b 8 c c c a (33.21) (44.91) (73.40) (100.0) (9.49) (12.33) (20.91) (-6.08) 2566.7±128.2 2083.3±64.1c 2016.7±71.5c 1683.3±70.3c 1350.0±57.7c 1066.7±44.1c 425.0±21.4d 0.0±0.0b 12 a (15.25) (17.12) (29.62) (43.36) (55.09) (81.91) (100.0) (-4.05) Amomum subulatum - - 0.5 g kg-1 1 g kg-1 3 g kg-1 0.5 g kg-1 1 g kg-1 3 g kg-1 0 g kg-1 7.5 mg kg-1 1241.7±101.2 0 1208.3±85.1a 1191.7±82.1a 1175.0±79.3a 1175.0±79.3a 1150.0±74.2a 1141.7±67.6a 1216.7±91.9a a 1050.0±71.9a 1150.0±88.5a 841.7±37.4b 1016.7±80.3a 833.3±69.1b 683.3±44.1b 1316.7±88.2a 75.0±17.1b 4 b (4.82) (28.37) (13.47) (27.54) (40.15) (-8.22) (93.96) (11.89) 1391.7±101.2 1058.3±79.0a 966.7±72.7ab 625.0±21.4c 741.7±53.9b 616.7±67.9c 433.3±21.1c 0.0±0.0b 8 a (12.41) (18.88) (46.81) (36.88) (46.37) (62.05) (100.0) (-14.38) 975.0±69.2a 866.7±74.9b 500.0±22.4c 641.7±47.3b 466.7±42.2c 291.7±8.3d 1475.0±94.6a 0.0±0.0b 12 (19.31) (27.27) (57.45) (45.39) (59.42) (74.45) (-21.23) (100.0) Acacia Nilotica bark - - 1 g kg-1 4 g kg-1 8 g kg-1 1 g kg-1 4 g kg-1 8 g kg-1 0 g kg-1 7.5 mg kg-1 3666.7±158.5 3616.7±151.5 3583.3±142.4 3550.0±160.7 3516.7±166.2 3483.3±158.0 3683.3±147.0 3683.3±147.0 0 a a a a a a a a 3600.0±153.8 3483.3±151.5 3158.3±122.1 2900.0±154.4 2425.0±116.0 3800.0±155.5 1841.7±79.0b 58.3±8.3b 4 a a b b b a (47.13) (98.42) (1.82) (3.69) (11.86) (18.31) (31.04) (-3.17) 3416.7±150.4 3358.3±151.9 2825.0±114.6 2383.3±132.1 3833.3±144.2 1758.3±83.1c 1191.7±52.3c 8.3±8.3b 8 a a b c a (50.00) (65.79) (99.77) (6.82) (7.14) (21.16) (32.86) (-4.07) 3325.0±141.3 3200.0±146.1 2450.0±103.3 1983.3±118.8 3925.0±145.9 1583.3±73.8c 975.0±44.3c 0.0±0.0b 12 a a c c a (54.98) (72.01) (100.0) (9.32) (11.52) (31.63) (44.13) (-6.56)

51 Table 29 Contd.. Day s Crude Powder Crude Aqueous Methanolic Extract Untreated Levamisole PT Acacia Nilotica leaves - - 1 g kg-1 4 g kg-1 8 g kg-1 1 g kg-1 4 g kg-1 8 g kg-1 0 g kg-1 7.5 mg kg-1 2766.7±108.5 2725.0±108.6 2716.7±108.5 2791.7±108.3 2800.0±106.5 0 2700.0±106.5a 2675.0±94.6a 2666.7±88.2a a a a a a 2708.3±109.1 2583.3±110.1 2400.0±114.8a 2091.7±120.7 2875.0±121.6 2441.7±92.6b 1808.3±63.8b 58.3±8.3b 4 a a b b a (10.12) (32.19) (97.92) (2.11) (5.20) (11.11) (21.81) (-2.98) 2625.0±109.4 2508.3±106.0 1041.7±170.5 2941.7±119.3 2141.7±71.2c 2166.7±111.6b 1616.7±47.7c 8.3±8.3b 8 a a c a (21.17) (19.75) (39.56) (99.70) (5.12) (7.95) (60.94) (-5.37) 2350.0±101.7 2975.0±127.0 2516.7±90.1a 1825.0±58.8d 1716.7±84.3c 1408.3±49.0c 975.0±35.9c 0.0±0.0b 12 a a (9.04) (32.82) (36.42) (47.35) (63.44) (100.0) (13.76) (-6.57) Vitex negundo - - 1 g kg-1 4 g kg-1 8 g kg-1 1 g kg-1 4 g kg-1 8 g kg-1 0 g kg-1 7.5 mg kg-1 0 1316.7±82.3a 1316.7±82.3a 1308.3±86.1a 1275.0±89.2a 1275.0±89.2a 1266.7±85.3a 1350.0±86.6a 1358.3±83.1a 1141.7±61.1a 1241.7±98.7a 1133.3±97.2a 1025.0±94.6b 1283.3±86.3a 1233.3±77.1a 1408.3±82.1a 50.0±0.0b 4 b (5.70) (13.93) (21.65) (-0.65) (3.27) (-4.32) (96.32) (9.87) 1175.0±103.9 1066.7±104.6 925.0±92.0b 1275.0±82.4a 1175.0±79.3a 966.7±66.7bc 1466.7±81.3a 0.0±0.0b 8 a a (29.30) (0.00) (7.84) (23.68) (-8.64) (100.0) (10.76) (18.99) 1125.0±103.9 991.7±106.8a 775.0±71.6b 1241.7±79.0a 1116.7±82.3a 858.3±56.9c 1516.7±72.7a 0.0±0.0b 12 a (24.68) (40.76) (2.61) (12.42) (32.24) (-12.35) (100.0) (14.56) Arundo donax - - 1 g kg-1 4 g kg-1 8 g kg-1 1 g kg-1 4 g kg-1 8 g kg-1 0 g kg-1 7.5 mg kg-1 0 1708.3±83.1a 1708.3±83.1a 1683.3±79.2a 1683.3±79.2a 1683.3±79.2a 1666.7±80.3a 1741.7±76.8a 1766.7±76.0a 1591.7±75.7a 1475.0±69.2a 1425.0±71.6b 1308.3±67.6b 1641.7±84.1a 1591.7±83.1a 1800.0±82.7a 50.0±0.0b 4 b b (16.58) (22.28) (2.47) (5.44) (-3.35) (97.17) (6.83) (11.50) 1491.7±66.3a 1308.3±63.8b 1208.3±45.5c 1091.7±45.5c 1600.0±76.4a 1508.3±83.1a 1841.7±87.9a 16.7±16.7b 8 b c (29.27) (35.15) (4.95) (10.40) (-5.74) (99.05) (12.68) (21.50) 1391.7±62.5b 1058.3±41.7c 833.3±38.0d 1533.3±79.2a 1400.0±76.4a 1141.7±67.6c 1883.3±81.3a 16.7±16.7b 12 (18.53) (38.05) (50.50) (8.91) (16.83) (31.50) (-8.13) (99.05) PT=Post-treatment; Means marked with the same letter in a column do not differ significantly at P ≥ 0.05; values in parenthesis indicate % reduction in EPG Table 30: Efficacy ranking based on reduction in fecal egg counts as on day 12 post- treatment (PT) of crude powder and crude methanol extract of different plants Plant % Reduction with Efficacy % Reduction with Efficacy crude powder (CP) ranking of CP crude methanol ranking of extract (CAME) CAME Amomum subulatum @ 3 g kg-1 57.5 1 74.5 3 Arundo donax @ 8 g kg-1 50.5 2 31.5 7 Ferula assa-foetida @ 1 g kg-1 41.2 3 87.6 1 Vitex negundo @ 8 g kg-1 40.8 4 32.3 6 Acacia nilotica leaves @ 8 g kg-1 32.9 5 63.5 5 Acacia nilotica bark @ 8 g kg-1 31.6 6 72.0 4 Areca catechu @ 1 g kg-1 29.6 7 81.9 2

52 Table 31: Ranking of efficacy of different CAME/fractions of plants based on three tests employed in the study Plant Ranking of Efficacy of Different CAME/Fractions of Plants in Different Evaluation Tests AMA* EHT** FECRT*** Ferula assa-foetida CAME 1 (10.00±0.000a) 4 (16.9126) 1 (87.6%) Ferula assa-foetida Chloroform fraction 1 (10.00±0.000a) 1 (06.9698) - Ferula assa-foetida Ethyl acetate fraction 1 (10.00±0.000a) 2 (10.6419) - Acacia nilotica bark CAME 1 (10.00±0.000a) - 4 (72.0%) Amomum subulatum Ethyl acetate fraction 1 (10.00±0.000a) 6 (18.6102) - Acacia nilotica leaves Ethyl acetate fraction 2 (09.67±0.333a) - - Areca catechue CAME 3 (09.00±0.577ab) - 2 (81.9%) Acacia nilotica bark Ethyl acetate fraction 4 (08.33±0.882bc) - - Amomum subulatum Chloroform fraction 5 (07.67±0.333bc) 5 (18.1413) - Amomum subulatum CAME 6 (07.33±0.667bc) 3 (13.1872) 3 (74.5%) Vitex negundo CAME 7 (07.00±0.577c) - 11 (32.3%) Acacia nilotica bark Chloroform fraction 9 (06.67±0.882bc) - - Areca catechue Ethyl acetate fraction 9 (06.67±0.882bc) - - Ferula assa-foetida Petroleum spirit 10 (06.33±0.333b) - - Amomum subulatum aqueous fraction - 7 (20.4178) - Ferula assa-foetida aqueous fraction - 8 (29.6247) - Vitex negundo aqueous fraction - 9 (38.7527) - Areca catechu aqueous fraction - 10 (38.9270) - Acacia nilotica leaves CAME - - 5 (63.5%) Arundo donax CAME - - 13 (31.5%) Amomum subulatum CP - - 6 (57.5%) Arundo donax CP - - 7 (50.5%) Ferula assa-foetida CP - - 8 (41.2%) Vitex negundo CP - - 9 (40.8%) Acacia nilotica leaves CP - - 10 (32.9%) Acacia nilotica bark CP - - 12 (31.6%) Areca catechu CP - - 14 (29.6%) Note: Values in the parenthesis indicate respective efficacy of the tests; whereas, numerical from 1-14 indicate efficacy ranking; *Results of AMA (Adult Motility Assay) given as number of dead worms at dose rate 50 mg mL-1 at 10 hours post-exposure; **Results of EHT (Egg Hatch Test) indicating LC50 values; ***Results of FECRT (Fecal Egg Count Reduction Test) given as % reduction on Day 12 post treatment; CP= crude powder; CAME= crude aqueous methanol extract

53 Chapter # 5

Discussion As mentioned earlier, objectives of the present study were to (i) document the ethnoveterinary practices with particular reference to the antiparasitic practices in some selected parts of District Jhang (Pakistan), and (ii) validate the claims of local healers regarding anthelmintic activity of some plants. Results of the study have been discussed as under: 5.1. Documentation of ethno-veterinary practices Salient findings of the study on ethno-veterinary medicine (EVM) documentation revealed that (i) of the total number of EVM practices reported by the respondents, remedies for worm infestation had the maximum contribution (≈17%), (ii) 46 plants were documented for their use in EVM practices in Jhang (Punjab, Pakistan) indicating an important role of plants in the treatment of different diseases of livestock, (iii) there was wide diversity in usage and in combination of different plants for the treatment of different diseases, and (iv) there was wide variation in the dose, vehicle, form of plant used, mode of preparation and administration/application for the use of plants even among the EVM practices for the same disease/condition. Though, it was hard for the herdsmen to describe the exact disease conditions, 26 ailments were reported from the study area afflicting livestock with the help of qualified veterinarians in the survey team. It could be because of the fact that in local language, diseases were named/described based on their symptoms. In contrast, etiological information is base of nomenclature of diseases in modern veterinary science (McCorkle, 1986; Mathias-Mundy and McCorkle, 1989). Of the total 26 livestock ailments documented in the area, mastitis was most frequently reported followed by worm infestation, red water, toxemia, prolapse, lochia retention, anestrous, anorexia, traumatic inflammation, indigestion, galactagogue, tympany, aglactia, allergy, chronic diarrhea, constipation, colic, external wounds, foot and mouth disease, non-specific jaundice, rheumatism, fever/foot rot, heat stroke/panting, hemorrhagic septicemia and pneumonia. Results revealed that number of EVM remedies were not essentially consistent with the number of entries of different diseases documented in the study. It was, however, interestingly noted that all the diseases of livestock documented in the area had one or more than one remedies prescribed by the traditional healers or self medicated by the farmers. Worm infestation and mastitis were the most frequently reported diseases in the study area. Accordingly, worm infestation was treated using the maximum variety of traditional remedies/plants followed by mastitis. It

54 appeared from the data that most of the diseases were due to poor management and nutritional deficiencies, which was typical of the orthodox rural culture. EVM has evolved through observation, trial and error, perfecting the techniques based on the experiences gathered through experimentation, and handling the resulting information down from one generation to the next (McCorkle, 1995). EVM mainly depends on the local knowledge of the people and this system of cure and treatment is commonly referred as “unani, folk, eastern or indigenous” medicines (Nadkarni, 1954). Therefore, the materials used in such indigenous medicinal systems are indigenous to the area, and therefore, have large variation from one region to the other. In the current study, of the 46 plants documented for their use in EVM, 33 were indigenous to the area. Availability of such a high number (n=33) of plants indicates pivotal role of indigenous plants in animal health care in the study area. The top 10 most frequently reported plants were Trachyspermum ammi, Capsicum annum, Vernonia anthelmintica, Foeniculum vulgare, Allium cepa, Piper nigrum, Ferula assa-foetida, Tamarindus indica, Amomum subulatum and Citrus limon. All these plants are commonly available as household spices/ítems in the villages and routinely used in human foods (cury, sauce, etc.). Great diversity was recorded in the usage of different plants. Maximum usage was recorded for Trachyspermum ammi used in eight EVM practices followed by Tamarindus indica, Cuminum cyminum, Capsicum annum, Vernonia anthelmintica, Amomum subulatum, Cannabis sativa, Citrus limon, Ferula assa-foetida and Linum usitatissimum used in 7, 6, 5, 5, 4, 4, 4, 4 and 4 EVM practices, respectively. Use of a particular plant for treatment of different diseases of both humans and animals is a common practice in traditional medicine system (Mathias-Mundy and McCorkle, 1989). Some of the plants documented during the present survey have other uses as well in the community. For example, some were used as food for humans and others were used as firewood. Generally, efforts for conservation for particular plant are usually improved if that plant has many different uses. Multiple use of a plant not only motivate the people to conserve species (Aguilar and Condit, 2001; Etkin, 2002) but also indicates its multiple actions. For example, use of Trachyspermum ammi in anestrus, colic, constipation, fever, indigestion, as lochia remover, tympany and worm infestation. This suggests a wide variety of chemicals present in Trachyspermum ammi. It has been observed that different factors like geographical region, storage methods and time of plant collections influence the pharmacological actions of a plant. There was maximum usage of seeds (n=37) followed by fruits (n=23), leaves (n=22), bulb (n=4), latex and pepper corns (n=3 each), aerial parts, bark, flowers and roots (n=2 each), fruit peel, resin and rhizomes (n=1 each). Maximum usage of seeds, fruit and leaves of plants in

55 traditional phytomedicine has been reported since centuries and it may be attributed to the convenient availability of these parts as household items and empirical evidence of their efficacy. Jaggery (n=30), molasses (n=28) and sodium chloride (n=8) were the most frequently used vehicles/adjuncts in the treatment of different ailments in animals. Jaggery and molasses are the proven energizers and sodium chloride is effectively used in digestive and metabolic disorders indicating their refreshing/tonic effects. Mode of preparation and administration of different recipes documented in this study are also typical of using traditional medicine as a convenient medicine. For example, use of sodium bicarbonate, sodium chloride, ammonium chloride, molasses, as well as special methods, such as hanging locket in the neck. Use allopathic veterinary drugs and materials other than plants have also been recorded elsewhere. For example, in Nigeria soap is used by herders (Alawa et al., 2003), used engine oil in Kenya (Heffernan et al., 1996) and in Thailand iron rust, talc and sulphur is used (Mathias-Mundy and McCorkle, 1989). All the respondents were having good knowledge of phytotherapy and other ethnoveterinary medicinal practices for prevention, control and treatment of different diseases. But they were of the view that these indigenous practices are now being replaced by modern veterinary medicine due to establishment of new hospitals, free availability of modern drugs by local government and most importantly due to steady loss of medicinal plants in field. Majority of the respondents reported that they learned indigenous knowledge from their forefathers and from elders of their community. Actually, knowledge of EVM is typically transferred by word of mouth (Gueye, 1997), especially in Pakistan, where very little information is available in written form. A few reports are available on documentation of ethno-veterinary practices in international literature (Lans et al., 2000; Veigi et al., 2003; Bowman, 2006; Njorogue and Bussmann, 2006). There is, however, increasing focus on ethno pharmacology due to problems of drug resistance and residual problems. Naturally, therefore, documentation is the first step to this direction. Documentation of EVM has been focused on recognition of the variations in animal health beliefs, practices and experiences of different social groups. Recently, Jabbar et al. (2006) published an inventory of ethno-veterinary practices used for treatment of gastrointestinal helminthiasis in Muzaffar Garh (Pakistan) located about 200 km away from the present study area (Jhang). Though, some of the plants reported by Jabbar et al. (2006) have also been reported in the present study, yet there are large numbers which have not been documented from Jhang area. The use of different plants for treatment of livestock diseases in different geographical regions is well evident from literature review (Lans et al., 2000; Veigi et al., 2003; Bowman, 2006; Njorogue and Bussmann, 2006). This variation in use of plants used for

56 phytotherapy indicates the diversity in EVM prescriptions, which needs to be scientifically validated and communicated to other communities. This will not only help the farmers in developing countries (Gesler, 1991), but will also be helpful for livestock farmers of developed countries, particularly in organic farming system. Issues that need to be addressed in order to satisfy the veterinarians for adaptation of EVM are efficacy, safety, quality and standardization of doses. World Health Organization has developed the guidelines for validation and development of medicines for humans (WHO, 2000), same could be modified for animals. Some of the ethno veterinary practices have been scientifically validated for their use among animals. Different plants, which have been evaluated for their anthelmintic activity include: Cyperus rotundus (Girgune et al., 1978), Zingiber officinale (Iqbal et al., 2001; Iqbal et al., 2006a), Azadirachta indica (BOSTID, 1992; Costa et al., 2006), Mallotus philippinensis (Akhtar and Ahmad, 1992) and Nicotiana tabacum (Iqbal et al., 2006b). Azadirachta indica has also been scientifically validated for its use as acaricide (Abdel-Shafy and Zayed, 2002). 5.2. Evaluation of anthelmintic activity of selected plants In vitro (adult motility assay and egg hatch test) and in vivo (fecal egg count reduction test) tests were carried out to evaluate the anthelmintic activity of selected plants. Ferula assa- foetida was effective in all the three tests used for evaluation of its anthelmintic activity; whereas, Acacia nilotica bark and Amomum subulatum were more effective against adult worms compared with their ovicidal activity. Amomum subulatum, however, proved a better ovicidal than Acacia nilotica bark in having high LC50 compared with Amomum subulatum. Likewise, Acacia nilotica leaves and Areca catechue were better against adult worms compared with their ovicidal effects. Chloroform and ethyle acetate fractions appeared to have better anthelmintic effects against adult worms compared with their ovicidal activity. Like in vitro tests; Ferula assa-foetida, Acacia nilotica bark, Amomum subulatum, Acacia nilotica leaves and Areca catechue, were among the top five most effective plants in vivo studies. Crude aqueous methanol extracts (CAME) of plants proved superior in anthelmintic effects compared with crude powder (CP) in vivo studies. The serial concentrations of extracts applied in vitro were about 3 to 90 times higher compared with the reference drug (levamisole) in adult motility assay and about 480 times higher compared with the reference drug (albendazole) in egg hatch assay. These concentrations have been selected based on our observations for the last about 07 years. Results of in vitro tests may not always correspond to those of in vivo studies but it is generally accepted that in vitro tests are good for preliminary screening of plants for their

57 anthelmintic efficacy. The authenticity of the in vitro and in vivo tests used for evaluation of the anthelmintic activity in the light of current results and available literature is discussed below: 5.2.1. Tests used for evaluation of the anthelmintic activity 5.2.1.1. Egg Hatch Test (EHT) The egg hatch test (EHT) was originally developed by Le Jambre (1976) for the detection of BZ resistance in livestock helminths. Later, it was adopted by World Association for the Advancement of Veterinary Parasitology after its standardization (Coles et al., 1992). It is based on the ovicidal activity of BZ. This test has, however, also been used for screening of plants and/or other compounds for their anthelmintic activity (Molan et al., 1999; Molan et al., 2000; Waghorn and Molan, 2001; Molan et al., 2002; Min et al., 2004). Reliable results can only be obtained if eggs are isolated from freshly collected feces (within 3 h of being shed). This is because of a false positive result due to development of eggs beyond the ventral indentation stage leading to embryonation (Le Jambre, 1976; Weston et al., 1984; Riou et al., 2005). Hunt and Taylor (1989) recommended the anaerobic storage of fecal sample where collection of eggs is not possible immediately after shedding of eggs. Anaerobic storage of fecal samples prevents the hatching of eggs and does not influence the results, at least for major gastrointestinal (GI) helminths of small ruminants. In this study, EHT was employed on H. contortus eggs using different concentrations of crude aqueous methanol extracts and its ethyle acetate, petroleum spirit, chloroform and aqueous fractions of different plants and benzimidazole (control). EHT was found useful in obtaining reliable data as evident from the varying efficacies (LC50) and dose-dependent effects of different extracts and fractions of plants screened in this study. Therefore, reliability of EHT as a drug/plant screening assay was in support of the earlier workers (Molan et al., 1999; Molan et al., 2000; Waghorn and Molan, 2001; Molan et al., 2002; Min et al., 2004). 5.2.1.2. Adult Motility Assay (AMA) Instead of all the advancement in the field of science of technology, it is not yet possible to maintain the in vitro culture of the H. contortus, though maturation of larvae to egg-laying adults has been reported by Stringfellow (1986). As adult stage of H. contortus is target of anthelmintic drugs, it would be most desirable to be able to determine the intrinsic potency of anthelmintics against them. At present, system available for maintaining the adult H. contortus in culture, after isolation from abomasums of sheep, is plagued by a continuous drop in viability of the worms with passage of time, which makes it difficult to interpret the results of bioassay. Adult motility assay (AMA) is, however, the most convenient test used for assaying the anthelmintic activity of drugs/plants/plant-products. In AMA, worms are exposed to varying

58 concentrations of drugs and observed for their inhibited motility and/or mortality at different intervals. To evaluate the anthelmintic activity of plant parts/extracts/oils different types of worms like; earthworm, Pheretima posthuma (Siddiqui and Garg, 1990), hookworms, H. contortus, tapeworms, A. lumbricoides, Teladorsagia cicumcincta and Trichostrongylus (T.) colubriformis (Sharma et al., 1971; Kalesaraj, 1974, 1975; Agarwal et al., 1979; Prakash et al., 1980; Paolini et al., 2003a; Hounzangbe-Adote et al., 2005) are being used. Toxic substances produce irritation/agitation to earthworm and consequently worm withdraw from the poisonous area. By virtue of this effect, wormicidal drugs expel the worms from their host, if the concentration of drug is not high enough to kill the worm (Sollmann, 1918). In the present study, adult H. contortus were used in adult motility assay and that proved to be a good test worm, due to its longer survival in phosphate buffer saline. Due to longer survival of worm in PBS, more observations were recorded during the bioassay. Adult motility assay is a simple and economical test. Worms required to evaluate the anthelmintic activity of a drug/plant extract can easily be collected from few animals. Furthermore, very little quantity of plant extract/chemical is required to prepare the test concentrations. Theoretically, AMA can be used for screening the anthelmintic activity of candidate drugs/compounds. As intestinal worms live in the lumen of intestine, anthelmintics administered through oral route reach at the site of predilection of the worms without much alteration in their chemical composition. However, it is true that a single test cannot be used to demonstrate the anthelmintic activity of candidate drug/compound with 100% confidence. But as a compromise between time, expense and labor the AMA used in the current study is good. 5.2.1.3. Fecal egg count reduction test (FECRT) Fecal egg count reduction test is used for the diagnosis of anthelmintic resistance, in vivo. In the present study, FECRT was used to check the anthelmintic activity of different plant extracts, with increased number of observations. In FECRT, pre- and post-treatment EPG values are used to calculate the percent decrease in egg count due to a particular drug (Boersema, 1983; Presidente, 1985). In the present study, McMaster egg counting method (Urquhart et al., 2003) was used to check the EPG, before and after the treatment, in different groups. A positive and negative control group was also included in the study to monitor the natural change in EPG during the study period. EPG only represent the mature worms parasitizing the host. FECRT may not estimate the efficacy of a candidate anthelmintic correctly as EPG does not correlate well with actual worm burden, it only represent the sexually mature population. Moreover, if the interval between treatments is less than 10 days, egg production may be suppressed leading to an overestimation of anthelmintic efficacy (Hotson et al., 1970; Martin et al., 1985). Therefore,

59 Coles et al. (1992) recommended that observations should be taken for a period of more than 12 days post treatment. FECRT can also take the worker to false negative or false positive (Jackson, 1993; Grimshaw et al., 1996) due to different developmental stages of the parasite, but this should not be a limitation in preliminary screening procedures. In vivo studies are usually terminated by the necropsies of animals in order to obtain information on the effect of the plants on the worm number and on the female fertility. Unfortunately, however, we were short of budget and could not afford to purchase the animals for slaughter. In conclusion, application of FECRT may be useful for preliminary in vivo testing of drugs, which can also be combined with copro-cultures to measure the effect against individual worm species. Copro-cultures were not carried out in this study due to some technical limitations. It was, however, deemed sufficient for preliminary screening of plants. The test was found useful as evident from the graded dose response recorded for the plants used as crude aqueous methanol extract and its fractions. 5.2.2. Anthelmintic activity of the selected plants Initially crude powders of plant parts were used to test their biological activities. Then aqueous extracts were used and now use of different solvents for making plant fractions helps to identify the plant fraction having better activity. Nature has gifted plants with a lot of chemical compounds having different properties. These compounds have different affinities for different solvents according to difference in polarities. Each fraction contains different chemical compounds due to different solvent used for extraction process. These active compounds in the fractions are responsible for the biological activities. Evaluation of efficacy of different fractions of botanicals may, therefore, help in discovery of new anthelmintic compounds. Results revealed that Ferula assa-foetida was effective in all the three tests used for evaluation of its anthelmintic activity; whereas, Acacia nilotica bark and Amomum subulatum were more effective against adult worms compared with their ovicidal activity. Amomum -1 subulatum, however, proved a better ovicidal (LC50=13.1872 µg mL ) than Acacia nilotica bark -1 (LC50= 201.0032 µg mL ). Likewise, Acacia nilotica leaves and Areca catechue were better against adult worms compared with their ovicidal effects. Chloroform and ethyle acetate fractions appeared to have better anthelmintic effects against adult worms compared with their ovicidal activity. Like in vitro tests; Ferula assa-foetida, Acacia nilotica bark, Amomum subulatum, Acacia nilotica leaves and Areca catechue, were among the top five most effective plants in in vivo studies as well. Crude aqueous methanol extracts (CAME) of plants proved superior in anthelmintic effects compared with crude powder (CP) in in vivo studies. A wide

60 variation was recorded in the anthelmintic effects among different plants/fractions as far as the intensity and dose dependent effects were concerned. Anthelmintic activity of plants evaluated in this study, traditional uses/pharmacological activities (particularly antimicrobial) and phytochemicals of the considered plants are given as under without much discussion on the mechanism of action except of those already reported. General discussion on the mechanism of action of various groups of phytochemicals as antimicrobials and their resembling possible actions to exert anthelmintic effects are given in section 5.3. 5.2.2.1. Ferula assa-foetida In adult motility assay, crude aqueous methanol extracts of Ferula assa-foetida @ 12.5- 50 mg mL-1 caused 100% mortality of H. contortus by 10 hours post-exposure. In EHT, Ferula -1 assa-foetida was the second highly ovicidal plant with LC50 = 16.9126 µg mL ; whereas, crude aqueous methanol extract of Ferula assa-foetida resulted in maximum reduction in EPG (87.6%) in sheep naturally infected with gastrointestinal nematodes. Ferula assa-foetida belongs to family Umbelliferae, which grows wild in Kashmir, Iran and Afghanistan. This family is a rich source of gum-resin (Fernch, 1971). It has an unpleasant smell, is herbaceous and perennial and grows up to 2 m high (Kapoor, 1990). The part used is an oleogum resin, obtained by incision from the root, and called asafoetida (Kapoor, 1990). The trading form is either the pure resin or so-called compounded asafoetida which is a fine powder consisting of more than 50% of rice flour and gum arabic to prevent lumping. The advantage of the compounded form (“hing”) is that it is easier to dose. Hing has very strong smell, rather repugnant, remotely similar to (not altogether fresh) garlic. Taste and smell of “hing” are due to sulfur containing compounds. Several fractions such as gum fraction (25%, including glucose, galactose, l-arabinose, rhamnose and glucuronic acid), resin (40–64%, which contains ferulic acid esters (60%), free ferulic acid (1.3%), coumarin derivatives (e.g. umbelliferone), volatile oils (3–17%) including sulphur-containing compounds, and various monoterpenes have been isolated from this plant (Kajimoto, 1989). Glucuronic acid, galactose, arabinose and rhamnose have been isolated from the gum (Kapoor, 1990). Disulfides as well as symmetric tri- and tetrasulfides have been isolated from Ferula assa-foetida (Rajanikanth et al., 1984). Umbelliferone, the farnesiferoles A, B and C, ferulic acid (Caglioti et al., 1958, 1959), and the cumarin derivatives foetidin and kamolonol are also present in Ferula assa-foetida (Hofer et al., 1984). Dried Ferula assa-foetida has been reported (Rajanikanth et al., 1984) to consist mostly of a resin (25 to 60% of the total mass, 60% of which are esters of ferula acid) and a complex carbohydrate part (25 to 30%). The essential

61 oil (10%) contains a wealth of sulfur compounds, mainly (R)-2-butyl-1-propenyl disulphide (50%), 1-(1-methylthiopropyl) 1-propenyl disulphide and 2-butyl-3-methylthioallyl disulphide. Furthermore, di-2-butyl trisulphide, 2-butyl methyl trisulphide, di-2-butyl disulphide and even di-2-butyl tetrasulphide have been found (Rajanikanth et al., 1984). In Iranian traditional medicine, Ferula assa-foetida gum extract has been used as a remedy for abdominal pain, constipation, and diarrhea and as an antihelminthic (Fernch, 1971). It is also considered to be a sedative, carminative, antispasmodic, diuretic, anthelmintic, emmenagogue, an expectorant, and an aphrodisiac (Eigner and Scholz, 1990). Assa-foetida produces slight inhibition of the growth of Staphylococcus aureus and Shigella sonnei, and some of the sulfur compounds show pesticidal activity. Higher doses taken orally may cause diarrhea, meteorism, headaches, dizziness and enhanced libido (Kapoor, 1990). Relaxant compounds in Ferula assa-foetida gum extract have been reported to interfere with a variety of muscarinic, adrenergic and histaminic receptor activities or with the mobilization of calcium ions required for smooth muscle contraction non-specifically (Fatehi et al., 2004). 5.2.2.2. Amomum subulatum In adult motility assay, 73.3% mortality of H. contortus was recorded by 10 hours post- exposure with crude aqueous methanol extracts of Amomum subulatum @ 50 mg mL-1. In EHT, -1 Amomum subulatum was the most effective ovicidal plant with LC50 = 13.1872 µg mL ; whereas, crude aqueous methanol extract of Amomum subulatum resulted in 74.5% reduction in EPG. Amomum subulatum fruit, commonly known as ‘Heel kalan’ or ‘Bari Ilaichi’ is used as spice throughout the world. In Unani system of medicine, Amomum subulatum is frequently prescribed in the treatment of gastrointestinal disorders and used as stomachic, digestive, antiemetic and carminative (Avicenna, 1912; Ibn Baitar, 1969; Ibn Rushd, 1980). Its seeds are used as flavoring spices, cardiac tonic, expectorant and diuretic. It is also used in anorexia, dyspepsia, hyperacidity, dysentery, skin diseases, wounds, ulcers, cardiac debility, liver congestion, cough, fever and gonorrhea (Sharma et al., 2002). It has also been claimed effective in liver disorders (Warrier et al., 1994) having hepatoprotective effects (Parmar et al., 2009). In animals, Amomum subulatum has been reported to be used for genital prolapse and anestrous (Dilshad et al., 2008), digestive disorders and anhidrosis (Muhammad et al., 2005). Phytochemical studies carried out on the fruits of Amomum subulatum revealed the presence of essential oils (Lawrence, 1970; Anonymous, 1985), anthocyanins (Lakshmi and Chauhan, 1976), aurone (Lakshmi and Chauhan, 1977), chalcone and a flavanone (Rao et al., 1976). Lawrence (1970) has reported 3% essential oil dominated by 1,8-cineol (more than 70%),

62 and variable amounts of limonene, terpinene, terpineol, terpinyl acetate and sabinene from seeds of Amomum subulatum. The essential oil isolated from the fruits of Amomum subulatum showed antifungal activity (Jain and Aggarwal, 1978; Mishra and Dubey, 1990). Phytoconstituents of Amomum subulatum like flavonoids, terpenoids, glycosides and volatile oils (Hikino et al., 1986; Defeudis et al., 2003; Takeoka and Dao, 2003; Quyang et al., 2003) are well known for their antioxidant and hepatoprotective activities. Jafri et al. (2001) has reported antiulcerogenic effects of fruits of Amomum subulatum, i.e., large cardamom. This may be due to direct protective effect of Amomum subulatum on gastric mucosal barrier and decreased gastric motility caused by essential oil and petroleum ether fractions suggesting gastroprotective action of the plant (Jafri et al., 2001). It is likely that phenolic compounds (flavanones, aurones or anthocyanins) present in Amomum subulatum, may be responsible for gastro protection (Rao et al., 1976). Bonjar (2004) has also reported antibacterial activity of Amomum subulatum. 5.2.2.3. Acacia nilotica In adult motility assay, 100 and 60% mortality of H. contortus was recorded by 10 hours post-exposure with crude aqueous methanol extracts of Acacia nilotica bark and leaves @ 50 mg mL-1, respectively. In EHT, Acacia nilotica bark and leaves exhibited weak ovicidal activity -1 with 201 and 769.2 µg mL LC50, respectively; whereas, crude aqueous methanol extract of Acacia nilotica bark and leaves resulted in 72 and 63.5% reduction in EPG. Acacia spp. are spiny shrubs or small trees, preferring sandy or sterile regions, with the climate dry during the greater part of the year. All the gum-yielding Acacias exhibit the same habit and general appearance, differing only in technical characters. Gum Acacia is a demulcent and serves by the viscidity of its solution to cover and sheath inflamed surfaces. Acacia nilotica is used as traditional forage for livestock. The leaves of Acacia nilotica are commonly fed to goats and sheep besides grazing (Maharaj and Dwivedi, 2002). Fruits of Acacia nilotica have been reported for their anthelmintic (Bachaya et al., 2009) and antifungal (Umalkar et al., 1977) activities. The present results are, however, in contrast to those of Kahiya et al. (2003) who evaluated Acacia nilotica leaves against artificial infection of Haemonchus contortus in goats and found to be ineffective in reducing the fecal egg count. These differences may be attributed to the actual leave content eaten up by the goats in the studies of Kahiya et al. (2003) and the dose of CAME given to sheep in this study. Major phytochemicals in Acacia spp. are flavonoid and tannins (Tindale and Roux, 1969; Malan and Roux, 1975; Devi and Prasad, 1991), cyanogenic glucosides (Secor et al., 1976), free amino acids (Evans et al., 1977; Evans and Bell, 1979), acacipetalin (Seigler et al.,

63 1978), labdane diterpenes (Forster et al., 1985), and proanthocyanidins and other phenolics (Dube, 1993). These phytochemicals are known for their antimicrobial activity (Cowan, 1999) and phenolics in particular may have their application as an anthelmintic. 5.2.2.4. Areca catechu In adult motility assay, 90% mortality of H. contortus was recorded by 10 hours post- exposure with crude aqueous methanol extracts of Arecha catechue @ 50 mg mL-1. In EHT, -1 Arecha catechue exhibited potent ovicidal activity with LC50 = 38.9 µg mL ; whereas, crude aqueous methanol extract of Arecha catechue resulted in 81.9% reduction in EPG in sheep naturally infected with gastrointestinal nematodes. Areca nuts are traditionally used as an anthelmintic (Raghavan and Baruah, 1958). The nut was once incorporated in the British pharmacopoeia as an anthelmintic to fight tapeworm and roundworm infections. Full descriptions of the pharmacology of betel are found in the studies of Arjungi (1976) and Mujumdar et al. (1979). Phytochemical studies on Areca catechue nuts have revealed the presence of tannin, along with gallic acid, a fixed oil gum, a small amount of volatile oil, lignin, and various saline substances (Huang, 1992), alkaloids including arecoline, arecaidine, guvacoline, guvacine, arecolidine and choline (Farnsworth, 1976; Chu, 2001), phenolic compounds, such as hydroxychavicol and safrole (Wang et al., 1997), and tannins, gallic acid, catechin, betasitosterol, gum and amino acids (Duke, 1992). Besides other activities (Bradley, 1979; Cawte, 1985; Kapoor, 1990; Lin et al., 2002), areca nut has also been reported to possess antiviral and antifungal properties (Huang, 1992). The anthelmintic activity of areca nuts recorded in the present study may be attributed to their pharmacological effects (Gilani and Ghayur, 2005) like stimulation of the relaxed bowel (Kapoor, 1990), cholinomimetic and acetylcholinesterase inhibitory effects (Gilani et al., 2004). Condensed tannins (CT) have been reported to exert direct or indirect biological effects on the control of gastrointestinal parasites. Niezen et al. (1995, 1998), Athanasiadou et al. (2000, 2000a, 2001), Butter et al. (2000) and Molan et al. (2002) have reported that direct effects might be mediated through CT nematode interactions, thereby affecting physiological functioning of parasites. CT also may react directly by interfering with parasite egg hatching and development to infective stage larvae. Molan et al. (2000) demonstrated that the CT extracted from Lotus pedunculatus, Lotus corniculatus, Hedysarum coronarium, and Onobrychus viciifolia forages reduced the rate of larval development (eggs to L3 larvae). Kahn and Diaz-Hernandez (2000) reported that CT extracted from various forages markedly decreased the viability of the larval stages of several nematodes in sheep and goats.

64 5.2.2.5. Vitex negundo In adult motility assay, 70% mortality of H. contortus was recorded by 10 hours post- exposure with crude aqueous methanol extracts of Vitex negundo @ 50 mg mL-1. In EHT, Vitex -1 negundo exhibited ovicidal activity with LC50 = 65.7 µg mL ; whereas, crude powder of Vitex negundo resulted in 40.8% reduction in EPG in sheep naturally infected with gastrointestinal nematodes. Vitex is used for treatment of different ailments like; depression, venereal diseases, malaria, asthma, allergy, wounds, skin diseases, snake bite and body pains (Neuwinger, 2000). Plants belonging to the genus Vitex (Yerbenaceae) are said to possess hormone-like properties [Sirai et al. (1962) cited by Subramanian and Misra (1979)]. An insect molting hormone, 20- hydroxy ecdysone was isolated from Vitex megapotamica [Rimpler and Shultz (1967) cited by Subramanian and Misra (1979)]. The earlier work on Vitex negundo reported the isolation of organic acids [Ghosh and Krishna (1936) and Quazi et al. (1973) cited by Subramanian and Misra (1979)] glucosides [Gupta and Sharma (1973) cited by Subramanian and Misra (1979)], essential oils, alkaloids [Kawa and Yamasita (1940) cited by Subramanian and Misra (1979); Basu and Singh, 1944; Basu and Lamsal, 1947] and flavonoids [Haensel et al. (1967) cited by Subramanian and Misra (1979); Banerjee et al., 1969] from leaves and β-sitosterol and n- alkanes [Gupta and Behari (1973) cited by Subramanian and Misra (1979)] from the seeds of the plant. The leaves of the plants were reported to produce an antiarthritic effect (Chaturvedi and Singh, 1965). Two new flavonoid glycosides were isolated from the stem bark of Vitex negundo by Subramanian and Misra (1979). Extracts of Vitex negundo possess anti- inflammatory, analgesic, hypouricaemic and anti-hyperglycemic activity (Dharmasiri et al., 2003; Villasenor and Lamadrid, 2006; Umamaheswari et al., 2007). Anti-oxidant and antiandrogenic properties were reported from the flavonoid-rich fraction of its seeds (Bhargava, 1989; Das et al., 2004). Vitex negundo has also been used as CNS depressant, for anti-histamine release and hepato-preventive purposes (Avadhoot and Rana, 1991; Nair et al., 1994; Gupta et al., 1999). Previous studies have already demonstrated significant analgesic activity of aqueous extract from Vitex negundo seeds (Zhong et al., 1996). 5.2.2.6. Arundo donax In adult motility assay, 56.7% mortality of H. contortus was recorded by 10 hours post- exposure with crude aqueous methanol extracts of Arundo donax @ 50 mg mL-1. In EHT, -1 Arundo donax exhibited ovicidal activity with LC50 = 200.1 µg mL ; whereas, crude powder of Arundo donax resulted in 50.5% reduction in EPG in sheep naturally infected with gastrointestinal nematodes. Arundo donax (Graminae) is a tall, stout perennial shrub, often

65 woody below, is widely distributed in India. A decoction of its rhizomes has been used in the Ayurvedic system of medicine as an emollient and diuretic and is said to stimulate menstrual discharge and to diminish the secretion of milk (Chopra et al., 1956). Arundo donax is also used as haemostatic and in toothache (Pieroni et al., 2002), pertussis and cystitis (Passalacqua et al., 2007). Ghosal et al. (1969) isolated five indole-3-alkylamine bases, viz., N,N- dimethyletryptamine, 5-methoxy-N-methyle-tryptamine, bufotenine, dehydrobufotenine and bufotenidine, from the rhizomes of Arundo donax. A defatted ethanolic extract of the rhizomes produced hypotensive and antispasmodic effects against histamine-, serotonine- and acetylecholine induced spasms. Bufotenidine showed three main pharmacological actions, viz., antiacetylecholine effect, histamine release and uterine stimulant. Ana et al. (2003) characterized lignin from the nodes and internodes of Arundo donax. 5.3. Phytochemicals as anthelmintics There is increasing evidence to support the hypothesis that plants are relatively high in bioactive secondary compounds and are thus likely to hold promise for drug discovery. Secondary compounds in weeds are important for a variety of ecological functions. Chief among these are allelopathy, where secondary compounds inhibit germination and growth of other plants; and, as chemical defense against herbivory (Harborne, 1993). At least 50 species of weeds have been shown to interfere with crops through allopathic secondary compounds (Putnam, 1994). Investigations into plant anti-herbivore defense are perhaps further developed. The two major anti-herbivory chemical defense strategies for plants are metabolically inactive immobile (or quantitative, as defined by Feeny (1976), defenses such as tannins and lignins that reduce digestibility; and mobile (or qualitative) defenses such as alkaloids, cardiac glycosides or terpenoids (Feeny, 1976; Coley et al., 1985). It is these latter types of compounds that are the basis for plant-derived pharmaceuticals. Ephemeral, successional or r-selected species (all common characteristics of weeds) tend to rely on these sorts of toxic chemical defenses (Rhoades and Cates, 1976; Abe and Higashi, 1991). Most of the plant derived chemicals are secondary metabolites, of which at least 12,000 have been isolated, a number estimated to be less than 10% of the total (Schultes, 1978). Nok et al. (1992, 1993) and Nok and Williams (1996) have discussed the active principles as well as the mechanisms of action of some plant extracts that are used in EVM. In these reports, appropriate dosages of the plant extracts required to suppress the growth of causative organisms of some diseases have also been given, which suggests the potential of traditional drugs in primary animal-health care. All the plants evaluated in the present study exhibited anthelmintic activity in one or the other tests. The ranking of different plants and fractions, however, varied in different tests. Some

66 of the top ranking plants exhibited anthelmintic activity in all the three tests used or two or even in one test. The variation in anthelmintic effects may primarily be attributed to the differences in the targets on the parasites to exert anthelmintic effects and chemistry of the plants. Although farmers have traditionally used plants for de-worming animals (Waller et al., 2001; Githiori et al., 2004), much work remains to be done so that traditional knowledge can aid the development of plant-based anthelmintic products that yield consistent results. Samples from different geographical regions have produced variable results (Waller et al., 2001) as the synthesis of secondary plant products can be affected by environmental growing conditions. Several tropical legumes have also shown some promising results. Grazing of L. cuneata forage (50 g CTs kg−1) achieved remarkably high reductions (57–100%) in faecal egg counts (FECs), total faecal egg output and the numbers of parasitic nematodes (species of Haemonchus, Teladorsagia and Trichostrongylus) in goats (Min and Hart, 2003). High levels of dried A. karoo leaves, which contain ca 240 g CTs kg−1 (Dube and Ndlovu, 1995) also significantly reduced FECs and Haemonchus contortus Rud. worm burdens in goats (Kahiya et al., 2003). In comparison, A. nilotica leaves had hardly any effect on FECs despite very high concentrations (ca 400 g kg−1) of catechin gallate tannins (Reed et al., 1985; Self et al., 1986; Mueller-Harvey et al., 1987; Kahiya et al., 2003). However, weight gains on both browse diets were comparable to a commercial goat meal diet after 7 weeks, which illustrates just some of the complexities of this type of research(Kahiya et al., 2003). Feeding of Acacia polyacantha Willd. reduced the FECs of a mixed nematode population by approximately 30% and the Oesophagostomum columbianum Curtice worm burden by 13% in goats (Max et al., 2004). The authors also noted that quebracho extracts appeared to be much more anthelmintic than wattle (Acacia mearnsii De Wild) extracts in vivo. This could stem from either concentration or slight structural differences between the tannins in these commercial extracts (Hagerman and Butler, 1981; Foo et al., 1986; Mueller-Harvey, 2006; Brunet et al., 2008). Together, these differences can produce an infinite variety of chemical structures, which in turn affect the physical and biological properties of the CT. Brunet and Hoste (2006) have demonstrated that the number of free hydroxy groups of CT monomers is a key factor in interactions with parasite larvae and nematode may have different susceptibility to monomers depending on species, which might be related to the protein composition of sheaths. Tannin rich extracts have been reported to exert a dose dependant effect on exsheathment of Haemonchus larvae (Brunet et al., 2007). Phytochemicals of the six plants discussed above can be broadly divided into phenolics and polyphenols (simple phenols, phenolic acids, quinines, flavones, flavonoids, flavonols,

67 tannins, coumarins, etc.), terpenoids and essential oils, alkaloids, lectins and polypeptides, mixtures, other compounds, etc. These all phytochemicals have been considered as useful in antimicrobial therapy (reviewed by Cowan, 1999). The efficacy of different plants based on their traditional/empirical use is attributed to the compounds or chemical groups given above. For example, phenolics possess a wide spectrum of biochemical activities such as antioxidant, antimutagenic and anticarcinogenic properties, as well as the ability to modify gene expression (Tapiero et al., 2002; Nakamura et al., 2003). There is, however, variation in the chemistry of plants, which leads to differences in the efficacy even within same species of plants. It has been reported that plant genotype (Scalzo et al., 2005) and cultivation (Hakkinen and Torronen, 2000) affect total phenolic and flavonoid contents in fruit. The variation of phenolic compounds depends on many factors, such as degree of maturity at harvest, genetic differences, environmental conditions, development stages of the plant at harvesting, drying process and storage technique (Heiberg et al., 1992; Prior et al., 1998; Kalt et al., 1999; Deighton et al., 2000; Wang and Lin, 2000; Connor et al., 2002; Shahidi and Naczk, 2004). Moreover, the type of solvent can also influence the amount and spectrum of the active components in the final extract of a plant. 5.3.1. Targets of anthelmintics Decades of basic research have identified some useful targets, in part through research that discovered how known anthelmintics work (certifying their “receptors” as useful targets for anthelmintics; see Lacey, 1988; Geary et al., 1992; Martin, 1993). The known target sites are solely proteins and include ion channels (e.g. Tetrahydropyrimidines, Imidazothiazoles, Macrocyclic lactones, Piperazine), enzymes (e.g. Benzimidazoles), structural proteins and transport molecules (e.g. Salicylanilides, Chlorinated sulfonamides) (Kohler, 2001). The pharmacological activity of Benzimidazoles, imidazothiazoles, and macrocyclic lactones is based on their affinity for specific receptors located inside the target parasite: β-tubulin, acetylcholine-gated channels and glutamate-gated chloride channels, respectively (Mottier et al., 2006). In many parasitic stages of helminths, an essential component of their fermentative metabolism, is the fumarate reductase reaction (e.g. Cheah and Bryant, 1966; Kmetec and Bueding, 1961; Prichard and Schofield, 1968; Scheibel et al., 1968). It has been shown that the broad spectrum anthelmintic thiabendazole (Prichard, 1970) and its analog, cambendazole (Malkin and Camacho, 1972) inhibit the fumarate reductase system of H. contortus; whereas, Van Den Bossche and Janssen (1969) found that 1-tetramisole inhibited the fumarate reductase system of a number of other nematodes.

68 A prime target for anthelmintic attack is the energy-generating system itself, i.e., the respiratory metabolic pathway that consumes carbohydrate and generates ATP, with reduced organic acids as excretory products. A drug which causes a breakdown in the respiratory metabolic pathway, or its control mechanisms, will be effective (Behm and Bryant, 1979). Compounds that increase the utilization of ATP beyond the parasite's capacity to generate it, will also be effective, and can be said to be acting on the respiratory pathway indirectly. Many anthelmintics are known to affect some of these pathways, in particular the pathway from phosphoenolpyruvate to propionate. The enzymes involved are phosphoenolpyruvate carboxykinase, malate dehydrogenase, fumarase, fumarate reductase and a complex succinate decarboxylase pathway. Phosphoenolpyruvate carboxykinase and fumarate reductase from Moniezia expansa are inhibited by cambendazole and mebendazole in vitro (Rahman and Bryant, 1977). Malate dehydrogenase from Fasciola hepatica is inhibited by a range of fasciolicides in vitro and in vivo, and it has been suggested that the synthesis of ATP in this pathway is prevented by limiting the supply of malate to the mitochondria (Lwin and Probert, 1975). The targets to exert anthelmintic effects may differ in various parasite stages. Grady and Kotze (2004) while discussing assay used for mechanism based screening for anthelmintic effects stated that potential problem with whole organism screening is that the screens are most easily applied to the free-living stages of parasite species (examples: larval development assay, larval motility assay, and egg hatch assay) whilst the ultimate use of the anthelmintic will be directed at the parasitic stages. The neurotoxic drug effects may be similar in free-living and parasitic stages; whereas, the biochemistry and physiology of free-living and parasitic stages differ in many aspects relevant to potential anthelmintic targets or potential detoxification mechanisms; for example, changes in energy metabolism from aerobic to anaerobic during the transition from free-living to parasitic life stages (Komuniecki and Komuniecki, 1995), decrease in oxidative detoxification capability in parasitic stages compared to free-living (Kotze, 1997). Based on the mode of action, the currently in market anthelmintics can be divided into nicotinic agonists, acetylcholinesterase inhibitors, GABA agonists, GluCI potentiators, calcium permeability increasers, β-tubulin binders, proton ionophores, inhibitors of malate metabolism, inhibitors of phosphoglycerate kinase and mutase, inhibitors of arachidonic acid metabolism and stimulators of innate immunity. 5.3.2. Mechanism of action of phytochemicals Phenolics and polyphenols are toxic to the microorganisms in different ways (Cowan, 1999). Simple phenols and phenolic acids cause enzyme inhibition by the oxidized

69 compounds, possibly through reaction with sulfhydryl groups or through more nonspecific interactions with the proteins (Mason and Wasserman, 1987). Quinones are known to complex irreversibly with nucleophilic amino acids in proteins (Stern et al., 1996), often leading to inactivation of the protein and loss of function. Probable targets in the microbial cell are surface- exposed adhesins, cell wall polypeptides, and membrane-bound enzymes (Cowan, 1999). The antimicrobial activity of flavones, flavonoids, and flavonols is probably due to their ability to complex with extracellular and soluble proteins and to complex with bacterial cell walls, more lipophilic flavonoids may also disrupt microbial membranes (Tsuchiya, et al., 1996). Tannins have been reported to complex with polysaccharide (Ya et al., 1988). Condensed tannins have been determined to bind cell walls of ruminal bacteria, preventing growth and protease activity (Jones et al., 1994). At least two studies have shown tannins to be inhibitory to viral reverse transcriptases (Kaul et al., 1985; Nonaka et al., 1990). One of the molecular actions of tannins is to complex with proteins through so-called nonspecific forces such as hydrogen bonding and hydrophobic effects, as well as by covalent bond formation (Haslam, 1996; Stern et al., 1996). Thus, their mode of antimicrobial action may be related to their ability to inactivate microbial adhesins, enzymes, cell envelope transport proteins, etc. The mechanism of action of the antimicrobial activity of terpenoids and essential oils (Vishwakarma, 1990; Scortichini and Rossi, 1991; Kubo et al., 1992; Ahmed, et al., 1993; Habtemariam et al., 1993; Harrigan et al., 1993; Kubo et al., 1993; Rao et al., 1993; Ayafor et al., 1994; Fujioka and Kashiwada, 1994; Hasegawa et al., 1994; Pengsuparp et al., 1994; Tassou et al., 1995; Ghoshal et al., 1996; Sun et al., 1996; Taylor et al., 1996; Xu et al., 1996; Barre et al., 1997; Mendoza et al., 1997; Rana et al., 1997; Suresh et al., 1997; Amaral et al., 1998) is not fully understood but is speculated to involve membrane disruption by the lipophilic compounds. The antimicrobial effects of alkaloids (Ghoshal et al., 1996; Freiburghaus et al., 1996; Omulokoli et al., 1997) such as berberine and harmane (Hopp et al., 1976) are attributed to their ability to intercalate with DNA (Phillipson and O’Neill, 1987). The inhibitory effect of lectins and polypeptides on microorganisms (Balls et al., 1942) may be due to the formation of ion channels in the microbial membrane (Terras et al., 1993; Zhang and Lewis, 1997) or competitive inhibition of adhesion of microbial proteins to host polysaccharide receptors (Sharon and Ofek, 1986). The antimicrobial effects may also be exerted by some mixtures of chemicals of plants such as those found in latex and propolis, which may act synergistically. This was proved by Amoros et al. (1992) who demonstrated that flavone and flavonol components were active in isolation against HSV-1, multiple flavonoids incubated

70 simultaneously with the virus were more effective than single chemicals, a possible explanation of why propolis is more effective than its individual compounds. Like other phytochemicals, condensed tannins (CT) have also been focused as to their potential as anthelmintics. About 40 years ago, Taylor and Murant (1966) reported the use of CT to reduce the soil nematode populations. Hence, it was surmised that the CT may be able to affect nematodes in the gastrointestinal tract of sheep. Therefore, the use of CT for anthelmintic purposes in animals has been a focused area of research particularly during the last 10–15 years. There are numerous reports indicating direct or indirect anthelmintic effects of CT (Barry et al., 1986; Dobson et al., 1990; Coop and Holmes, 1996; Van Houtert and Sykes, 1996; Donaldson et al., 1997; Aerts et al., 1999; Kahn and Diaz-Hernandez, 2000; Athanasiadou et al., 2001; Niezen et al., 2002a; Iqbal et al., 2002). The inhibitory effects on egg hatching of T. colubriformis have been reported previously (Niezen et al., 2002a) after feeding tanniferous diets to lambs. Earlier, it has been shown that CT extracted from herbage can inhibit the migration of T. colubriformis L3 in in vitro assays (Molan et al., 2000) indicating that CT can be detrimental to both egg hatching and larval development. Iqbal et al. (2007) reported that 25–100 mg/mL concentrations of CT did not exert anthelmintic effects on adult H. contortus. As far as ascertained, anthelmintic effect of CT on adult worms has not so far been investigated in vitro. The larval development/viability assays have, however, indicated that larval development was not affected, but the viability of infective nematode larvae was adversely affected after exposure to different concentrations (0–10%) of Quebracho tannins (Athanasiadou et al., 2001). It was speculated that the anthelmintic activity of Quebracho extract against infective larvae may be due to capacity of tannin to bind to proteins, which resulted in reduced nutrient availability, and thus larvae starvation and death. Condensed tannins may also bind to the cuticle of larvae, which is high in glycoprotein (Thompson and Geary, 1995) and cause their death. The difference in the anthelmintic effects of CT on infective larvae (Athanasiadou et al., 2001) and adult H. contortus (Iqbal et al., 2007) could be attributed to the interspecific differences in susceptibility as demonstrated by Athanasiadou et al. (2001). It may also be attributed to the difference in stage of the parasite used. The adult worm may have become more resilient to CT compared with the larval form. The discussion on phytochemical groups having antimicrobial properties suggests that inspite of differences in the biology of bacteria, fungi, protozoa, and helminths, there are some common targets among them which can also be utilized by the compounds having anthelmintic activity. These may include inhibition of enzymes, complexing with proteins, polysaccharide, formation of ion channels, etc. Such targeted interventions may result in disturbing the normal

71 biochemical and physiological processes leading to starvation, structural changes, neuromuscular interruptions, and other effects on helminths. In fact, most of these are the known target sites for commonly used anthelmintics (Kohler, 2001; Mottier et al., 2006).

72 Chapter # 6 Summary The objectives of the present study were to (i) document the ethno-veterinary knowledge (EVM) with particular reference to the antiparasitic practices in some selected parts of District Jhang (Pakistan), and (ii) to validate the claims of local healers regarding anthelmintic activity of some plants. A total of 30 villages of district of Jhang were included in the study for documentation of EVM practices. Information was collected from the local experts/traditional healers following Rapid Rural Appraisal (RRA) and Participatory Rural Appraisal Techniques using questionnaires, interviews and focused group discussions. Seven of the 46 documented plants/parts of plant were selected for validation of their anthelmintic activity following standard parasitological procedures, i.e. Adult Motility Assay, Egg Hatch Test and Fecal Egg Count Reduction Test. Salient findings of the study on EVM documentation were that: (i) of the total number of EVM practices reported by the respondents, remedies for worm infestation had the maximum contribution (≈17%), (ii) 46 plants were documented for their use in EVM practices in Jhang (Punjab, Pakistan) indicating an important role of plants in the treatment of different diseases of livestock, (iii) there was wide variation in the dose, vehicle, form of plant used, mode of preparation and administration/application for the use of plants even among the EVM practices for the same disease/condition, (iv) there was wide diversity in usage and in combination of different plants for the treatment of different diseases. Studies on the anthelmintic activity revealed that Ferula assa-foetida was effective in all the three tests used for evaluation of its anthelmintic activity; whereas, Acacia nilotica bark and Amomum subulatum were more effective against adult worms compared with their ovicidal -1 activity. Amomum subulatum, however, proved a better ovicidal (LC50=13.2 µg mL ) than -1 Acacia nilotica bark (LC50= 201.0 µg mL ). Likewise, Acacia nilotica leaves and Areca catechue were better against adult worms compared with their ovicidal effects. Chloroform and ethyle acetate fractions appeared to have better anthelmintic effects against adult worms compared with their ovicidal activity. Like in vitro tests; Ferula assa-foetida, Acacia nilotica bark, Amomum subulatum, Acacia nilotica leaves and Areca catechue, were among the top five most effective plants in vivo studies. Crude aqueous methanol extracts (CAME) of plants proved superior in anthelmintic effects compared with crude powder (CP) in vivo studies. Based on this study, it is recommended that (i) documentation work may be expanded to other areas having rich cultural heritage and indigenous knowledge, (ii) all the plants used in

73 EVM may be subjected to standard scientific procedures for their validation, (iii) farmers may be communicated the results of present study on validation of anthelmintic activity so that they can make a better choice of local plants in view of their varying efficacy, and (iv) further controlled studies on large number of animals may be continued.

74 Chapter # 7

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